Method for simultaneous production of multiple proteins; vectors and cells for use therein

ABSTRACT

Described is the production of proteins in a host cell. More specifically, described are methods for improving expression of two or more proteins in a cell or host cell. The methods are suited for production of, for example, recombinant antibodies that can be used in pharmaceutical preparations or as diagnostic tools. In one embodiment, provided is a method for obtaining a cell that expresses two or more proteins comprising providing the cell with two or more protein expression units encoding two or more proteins, characterized in that at least two of the protein expression units comprise at least one STAR sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/013,031, filed Dec. 14, 2004, now U.S. Pat. No. 7,364,878,which is a continuation application of International Patent ApplicationNo. PCT/NL03/00432, filed Jun. 13, 2003, designating the United States,and published in English as International Patent Publication No. WO03/106684 on Dec. 24, 2003, which claims benefit, under the ParisConvention, of European Patent Application No. EP 02077350 filed Jun.14, 2002, the entirety of each of which are hereby incorporated byreference.

STATEMENT ACCORDING TO 37 C.F.R. §1.52(e)(5)—SEQUENCE LISTING SUBMITTEDON COMPACT DISC

Pursuant to 37 C.F.R. §1.52(e)(1)(iii), a compact disc containing anelectronic version of the Sequence Listing has been submittedconcomitant with this application, the contents of which are herebyincorporated by reference. A second compact disk is submitted and is anidentical copy of the first compact disc. The discs are labeled, “copy1” and “copy 2,” respectively, and each disc contains one file entitled“P60556PC00.txt” which is 502 KB, and created on Nov. 29, 2004.

TECHNICAL FIELD

The invention relates to the fields of biochemistry, molecular biology,pharmacology and diagnosis. More specifically, the present inventionrelates to the production of proteins in a host cell. Even morespecifically, the invention relates to a method for improving expressionof two or more proteins in a (host) cell. The method is suited forproduction of, for example, recombinant antibodies that can be used in apharmaceutical preparation or as a diagnostic tool.

BACKGROUND OF THE INVENTION

Proteins are produced in systems for a wide range of applications inbiology and biotechnology. These include research into cellular andmolecular function, production of proteins as biopharmaceuticals ordiagnostic reagents, and modification of the traits or phenotypes oflivestock and crops. Biopharmaceuticals are usually proteins that havean extracellular function, such as antibodies for immunotherapy orhormones or cytokines for eliciting a cellular response. Proteins withextracellular functions exit the cell via the secretory pathway, andundergo post-translational modifications during secretion. Themodifications (primarily glycosylation and disulfide bond formation) donot occur in bacteria. Moreover, the specific oligosaccharides attachedto proteins by glycosylating enzymes are species and cell-type specific.These considerations often limit the choice of host cells forheterologous protein production to eukaryotic cells (Kaufman, 2000). Forexpression of human therapeutic proteins, host cells such as bacteria,yeast, or plants may be inappropriate. Even the subtle differences inprotein glycosylation between rodents and human, for example, can besufficient to render proteins produced in rodent cells unacceptable fortherapeutic use (Sheeley et al., 1997). The consequences of improper(i.e., non-human) glycosylation include immunogenicity, reducedfunctional half-life, and loss of activity. This limits the choice ofhost cells further, to human cell lines or to cell lines such as ChineseHamster Ovary (CHO) cells, which may produce glycoproteins withhuman-like carbohydrate structures (Liu, 1992).

Some proteins of biotechnological interest are functional as multimers,i.e., they consist of two or more, possibly different, polypeptidechains in their biologically and/or biotechnologically active form.Examples include antibodies (Wright & Morrison, 1997), bonemorphogenetic proteins (Groeneveld & Burger, 2000), nuclear hormonereceptors (Aranda & Pascual, 2001), heterodimeric cell surface receptors(e.g., T cell receptors, (Chan & Mak, 1989)), integrins (Hynes, 1999),and the glycoprotein hormone family (chorionic gonadotrophin, pituitaryluteinizing hormone, follicle-stimulating hormone, andthyroid-stimulating hormone, (Thotakura & Blithe, 1995)). Production ofsuch multimeric proteins in heterologous systems is technicallydifficult due to a number of limitations of current expression systems.These limitations include (1) difficulties in isolating recombinantcells/cell lines that produce the monomer polypeptides at high levels(predictability and yield), (2) difficulties in attaining production ofthe monomeric polypeptides in stoichiometrically balanced proportions(Kaufman, 2000), and (3) declines in the levels of expression during theindustrial production cycle of the proteins (stability). These problemsare described in more detail below.

(1) Recombinant proteins such as antibodies that are used as therapeuticcompounds need to be produced in large quantities. The host cells usedfor recombinant protein production must be compatible with the scale ofthe industrial processes that are employed. Specifically, the transgene(or the gene encoding a protein of interest, the two terms are usedinterchangeably herein) expression system used for the heterologousprotein needs to be retained by the host cells in a stable and activeform during the growth phases of scale-up and production. This isachieved by integration of the transgene into the genome of the hostcell. However, creation of recombinant cell lines by conventional meansis a costly and inefficient process due to the unpredictability oftransgene expression among the recombinant host cells. Theunpredictability stems from the high likelihood that the transgene willbecome inactive due to gene silencing (McBurney et al., 2002). Usingconventional technologies, the proportion of recombinant host cells thatproduce one polypeptide at high levels ranges from 1-2%. In order toconstruct a cell line that produces two polypeptides at high levels, thetwo transgenes are generally integrated independently. If the twotransgenes are transfected simultaneously on two separate plasmids, theproportion of cells that will produce both polypeptides at high levelswill be the arithmetic product of the proportions for single transgenes.Therefore, the proportion of such recombinant cell lines ranges from onein 2,500 to one in 10,000. For multimeric proteins with three or moresubunits, the proportions decline further. These high-producing celllines must subsequently be identified and isolated from the rest of thepopulation. The methods required to screen for these rarehigh-expressing cell lines are time-consuming and expensive.

An alternative to simultaneous transfection of two transgene-bearingplasmids is sequential transfection. In this case the proportion ofhigh-yielding clones will be the sum of the proportions for singletransgenes, i.e., 2-4%. Sequential transfection however has (major)drawbacks, including high costs and poor stability. The high costsresult from various factors: in particular, the time and resourcesrequired for screening for high-expressing cell lines is doubled, sincehigh expression of each subunit must be screened for separately. Thepoor overall stability of host cells expressing two polypeptides is aconsequence of the inherent instability of each of the two transgenes.

(2) Production of multimeric proteins requires balanced levels oftranscriptional and translational expression of each of the polypeptidemonomers. Imbalanced expression of the monomers is wasteful of thecostly resources used in cell cultivation. Moreover, the imbalancedexpression of one monomer can have deleterious effects on the cell.These effects include (a) sequestration of cellular factors required forsecretion of the recombinant proteins (e.g., chaperones in theendoplasmic reticulum, (Chevet et al., 2001)), and (b) induction ofstress responses that result in reduced rates of growth and proteintranslation, or even in apoptosis (programmed cell death) (Pahl &Baeuerle, 1997, Patil & Walter, 2001). These deleterious effects lead tolosses in productivity and yield and to higher overhead costs.

(3) Silencing of transgene expression during prolonged host cellcultivation is a commonly observed phenomenon. In vertebrate cells itcan be caused by formation of heterochromatin at the transgene locus,which prevents transcription of the transgene. Transgene silencing isstochastic; it can occur shortly after integration of the transgene intothe genome, or only after a number of cell divisions. This results inheterogeneous cell populations after prolonged cultivation, in whichsome cells continue to express high levels of recombinant protein whileothers express low or undetectable levels of the protein (Martin &Whitelaw, 1996, McBurney et al., 2002). A cell line that is used forheterologous protein production is derived from a single cell, yet isoften scaled up to, and maintained for long periods at, cell densitiesin excess of ten million cells per milliliter in cultivators of 1,000liters or more. These large cell populations (10¹⁴-10¹⁶ cells) are proneto serious declines in productivity due to transgene silencing(Migliaccio et al., 2000, Strutzenberger et al., 1999).

The instability of expression of recombinant host cells is particularlysevere when transgene copy numbers are amplified in an attempt toincrease yields. Transgene amplification is achieved by including aselectable marker gene such as dihydrofolate reductase (DHFR) with thetransgene during integration. Increased concentrations of the selectionagent (in the case of DHFR, the drug methotrexate) select for cells thathave amplified the number of DHFR genes in the chromosome. Since thetransgene and DHFR are co-localized in the chromosome, the transgenecopy number increases too. This is correlated with an increase in theyield of the heterologous protein (Kaufman, 1990). However, the tandemrepeats of transgenes that result from amplification are highlysusceptible to silencing (Garrick et al., 1998, Kaufman, 1990, McBurneyet al., 2002). Silencing is often due to a decline in transgene copynumber after the selection agent is removed (Kaufman, 1990). Removal ofthe selection agent, however, is routine during industrialbiopharmaceutical production, for two reasons. First, cultivation ofcells at industrial scales in the presence of selection agents is noteconomically feasible, as the agents are expensive compounds. Second,and more importantly, concerns for product purity and safety precludemaintaining selection during a production cycle. Purifying a recombinantprotein and removing all traces of the selection agent is necessary ifthe protein is intended for pharmaceutical use. However, it istechnically difficult and prohibitively expensive to do so, anddemonstrating that this has been achieved is also difficult andexpensive. Therefore, amplification-based transgenic systems thatrequire continual presence of selection agents are disadvantageous.

Alternatively, silencing can be due to epigenetic effects on thetransgene tandem repeats, a phenomenon known as Repeat Induced GeneSilencing (RIGS) (Whitelaw et al., 2001). In these cases the copy numberof the transgene is stable, and silencing occurs due to changes in thechromatin structure of the transgenes (McBurney et al., 2002). Thepresence of a selection agent during cell cultivation may be unable toprevent silencing of the transgene transcription unit because transgeneexpression is independent of expression of the selectable marker. Thelack of a means to prevent RIGS in conventional transgenic systems thusresults in costly losses in productivity.

SUMMARY OF THE INVENTION

The problems associated with conventional transgene expressiontechnologies for protein production and more specifically for multimericprotein production demonstrate a need in the art for a system thatovercomes these problems. The present invention relates to a novelsystem for creating (host) cells/cell lines that efficiently express twoor more proteins, for example, two or more polypeptide monomers andoptionally produce functional multimeric proteins from them. Importantexamples of heterologous multimer proteins are recombinant antibodies.In one embodiment, the invention takes advantage of proprietary DNAelements that protect transgenes from silencing, termed STabilizingAnti-Repressor (STAR or STAR™; these terms will be used interchangeablyherein) elements, for the production of two or more proteins.

The invention also discloses a novel configuration of transcriptionaland translational elements and selectable marker genes. In oneembodiment, the invention uses antibiotic resistance genes and proteintranslation initiation sites with reduced translation efficiency (forexample, an Internal Ribosome Entry Site, IRES) in novel ways thatimprove heterologous protein expression. The combination of the STARelements and these other elements results in a system for obtaining acell which expresses two or more proteins that (1) predictably producesa high proportion of recombinant cell lines with high yields ofheterologous proteins, (2) exhibits balanced and proportional expressionof two or more polypeptide monomers which are constituents of amultimeric protein, and (3) creates recombinant cell lines with stableproductivity characteristics.

Therefore, the invention provides in one embodiment, a method forobtaining a cell which expresses two or more proteins comprisingproviding the cell with two or more protein expression units encodingthe two or more proteins, characterized in that at least two of theprotein expression units comprise at least one STAR sequence.

STAR-sequences can be identified (as disclosed, for example, in Example1 of EP 01202581.3) using a method of detecting, and optionallyselecting, a DNA sequence with a gene transcription-modulating quality,comprising providing a transcription system with a variety offragment-comprising vectors, the vectors comprising i) an element with agene-transcription repressing quality, and ii) a promoter directingtranscription of a reporter gene, the method further comprisingperforming a selection step in the transcription system in order toidentify the DNA sequence with the gene transcription modulatingquality. Preferably, the fragments are located between i) the elementwith a gene-transcription repressing quality, and ii) the promoterdirecting transcription of the reporter gene. RNA polymerase initiatesthe transcription process after binding to a specific sequence, calledthe promoter, that signals where RNA synthesis should begin. Amodulating quality can enhance transcription from the promoter in cis,in a given cell type and/or a given promoter. The same DNA sequence cancomprise an enhancing quality in one type of cell or with one type ofpromoter, whereas it can comprise another or no gene transcriptionmodulating quality in another cell or with another type of promoter.Transcription can be influenced through a direct effect of theregulatory element (or the protein(s) binding to it) on thetranscription of a particular promoter. Transcription can however, alsobe influenced by an indirect effect, for instance because the regulatoryelement affects the function of one or more other regulatory elements. Agene transcription modulating quality can also comprise a stable genetranscription quality. With stable is meant that the observedtranscription level is not significantly changed over at least 30 celldivisions. A stable quality is useful in situations wherein expressioncharacteristics should be predictable over many cell divisions. Typicalexamples are cell lines transfected with foreign genes. Other examplesare transgenic animals and plants and gene therapies. Very often,introduced expression cassettes function differently after increasingnumbers of cell divisions or plant or animal generations. Preferably, astable quality comprises a capacity to maintain gene transcription insubsequent generations of a transgenic plant or animal. Of course, ifexpression is inducible, this quality comprises the quality to maintaininducibility of expression in subsequent generations of a transgenicplant or animal. Frequently, expression levels drop dramatically withincreasing numbers of cell divisions. With the herein described methodfor identification of a DNA sequence with a gene transcriptionmodulating quality, it is possible to detect and optionally select a DNAsequence that is capable of at least in part preventing the dramaticdrop in transcription levels with increasing numbers of cell divisions.Preferably, the gene transcription modulating quality comprises a stablegene transcription quality. Strikingly, fragments comprising a DNAsequence with the stable gene transcription quality can be detected andoptionally selected with the method for identification of a DNA sequencewith a gene transcription modulating quality, in spite of the fact thatthis method does not necessarily measure long term stability oftranscription. Preferably, this gene transcription modulating qualitycomprises a stable gene transcription enhancing quality. It has beenobserved that incorporation of a DNA sequence with a gene transcriptionmodulating quality in an expression vector with a gene of interest,results in a higher level of transcription of the gene of interest, uponintegration of the expression vector in the genome of a cell andmoreover that the higher gene expression level is also more stable thanin the absence of the DNA sequence with a gene transcription modulatingquality.

In experiments designed to introduce a gene of interest into the genomeof a cell and to obtain expression of the gene of interest, thefollowing has been observed. If together with the gene of interest alsoa DNA sequence with a gene transcription modulating quality wasintroduced, more clones could be detected that expressed more than acertain amount of gene product of the gene of interest, than when theDNA sequence was not introduced together with the gene of interest. Thusan identified DNA sequence with gene transcription modulating qualityalso provides a method for increasing the number of cells expressingmore than a certain level of a gene product of a gene of interest uponproviding the gene of interest to the genome of the cells, comprisingproviding the cell with a DNA sequence comprising a gene transcriptionmodulating quality together with the gene of interest.

The chances of detecting a fragment with a gene transcription-modulatingquality vary with the source from which the fragments are derived.Typically, there is no prior knowledge of the presence or absence offragments with that quality. In those situations many fragments will notcomprise a DNA sequence with a gene transcription-modulating quality. Inthese situations a formal selection step for DNA sequences with thatquality is introduced. This is done by selection vectors comprising thesequence on the basis of a feature of a product of the reporter gene,that can be selected for or against. For instance, the gene product mayinduce fluorescence or a color deposit (e.g., green fluorescent proteinand derivatives, luciferase, or alkaline phosphatase) or conferantibiotic resistance or induce apoptosis and cell death.

A method for the identification of a DNA sequence with a genetranscription modulating quality is particularly suited for detectingand optionally selecting a DNA sequence comprising a genetranscription-enhancing quality. It has been observed that at least someof the selected DNA sequences, when incorporated into an expressionvector comprising a gene of interest, can dramatically increase genetranscription of the gene of interest in a host cell even when thevector does not comprise an element with a gene-transcription repressingquality. This gene transcription enhancing quality is very useful incell lines transfected with foreign genes or in transgenic animals andplants.

The transcription system can be a cell free in vitro transcriptionsystem. With the current expertise in automation such cell free systemscan be accurate and quick. However, the transcription system preferablycomprises host cells. Using host cells warrants that fragments aredetected and optionally selected with activity in cells.

An element with a gene transcription repressing quality will represstranscription from a promoter in the transcription system used.Repression does not have to lead to undetectable expression levels.Important is that the difference in expression levels in the absence orpresence of repression is detectable and optionally selectable.Preferably, gene-transcription repression in the vectors results ingene-transcription repressing chromatin. Preferably, DNA sequences canbe detected, and optionally selected that are capable of at least inpart counteracting the formation of gene-transcription repressingchromatin. In one aspect a DNA sequence capable of at least in partcounteracting the formation of gene-transcription repressing chromatincomprises a stable gene transcription quality. Preferably, the DNAsequence involved in gene-transcription repression is a DNA sequencethat is recognized by a protein complex and wherein the transcriptionsystem comprises the complex. Preferably, the complex comprises aheterochromatin-binding protein comprising HP1, a Polycomb-group (Pc-G)protein, a histone deacetylase activity or MeCP2 (methyl-CpG-bindingprotein). Many organisms comprise one or more of these proteins. Theseproteins frequently exhibit activity in other species as well. Thecomplex can thus also comprise proteins from two or more species. Thementioned set of known chromatin-associated protein complexes are ableto convey long-range repression over many base pairs. The complexes arealso involved in stably transferring the repressed status of genes todaughter cells upon cell division. Sequences selected in this way areable to convey long-range anti-repression over many base pairs (van derVlag et al., 2000).

The vector used can be any vector that is suitable for cloning DNA andthat can be used in a transcription system. When host cells are used itis preferred that the vector is an episomally replicating vector. Inthis way, effects due to different sites of integration of the vectorare avoided. DNA elements flanking the vector at the site of integrationcan have effects on the level of transcription of the promoter andthereby mimic effects of fragments comprising DNA sequences with a genetranscription modulating quality. In a preferred embodiment, the vectorcomprises a replication origin from the Epstein-Barr virus (EBV), OriP,and a nuclear antigen (EBNA-1). Such vectors are capable of replicatingin many types of eukaryotic cells and assemble into chromatin underappropriate conditions.

DNA sequences with gene transcription modulating quality can be obtainedfrom different sources, for example, from a plant or vertebrate, orderivatives thereof, or a synthetic DNA sequence or one constructed bymeans of genetic engineering. Preferably, the DNA sequence comprises asequence as depicted in Table 3 and/or SEQ ID NOs:1-119 and/or afunctional equivalent and/or a functional fragment thereof. SEQ IDNOs:1-119 comprise STAR1-STAR65 (SEQ ID NOS:1-65); sequences comprisingSTAR66 and testing set (SEQ ID NOS:66-84); and sequences comprisingArabidopsis STAR A1-A35 (SEQ ID NOS:85-119) (herein after SEQ IDNOs:1-119).

Several methods are available in the art to extract sequence identifiersfrom a family of DNA sequences sharing a certain common feature. Suchsequence identifiers can subsequently be used to identify sequences thatshare one or more identifiers. Sequences sharing such one or moreidentifiers are likely to be members of the same family of sequences,i.e., they are likely to share the common feature of the family. Herein,a large number of sequences comprising STAR activity (so-called STARsequences or STAR elements) were used to obtain sequence identifiers(patterns) which are characteristic for sequences comprising STARactivity. These patterns can be used to determine whether a testsequence is likely to contain STAR activity. A method for detecting thepresence of a STAR sequence within a nucleic acid sequence of about50-5000 base pairs is thus herein provided, comprising determining thefrequency of occurrence in the sequence of at least one sequence patternand determining that the frequency of occurrence is representative ofthe frequency of occurrence of at least one sequence pattern in at leastone sequence comprising a STAR sequence. In principle any method issuited for determining whether a sequence pattern is representative of aSTAR sequence. Many different methods are available in the art.Preferably, the step of determining that the occurrence isrepresentative of the frequency of occurrence of at least one sequencepattern in at least one sequence comprising a STAR sequence comprises,determining that the frequency of occurrence of at least one sequencepattern significantly differs between at least one STAR sequence and atleast one control sequence. In principle any significant difference isdiscriminative for the presence of a STAR sequence. However, in aparticularly preferred embodiment, the frequency of occurrence of atleast one sequence pattern is significantly higher in at least onesequence comprising a STAR sequence compared to at least one controlsequence.

As described above, a considerable number of sequences comprising a STARsequence have been identified herein. It is possible to use thesesequences to test how efficient a pattern is in discriminating between acontrol sequence and a sequence comprising a STAR sequence. Usingso-called discriminant analysis it is possible to determine on the basisof any set of STAR sequences in a species, the most optimaldiscriminative sequence patters or combination thereof. Thus,preferably, at least one of the patterns is selected on the basis ofoptimal discrimination between at least one sequence comprising a STARsequence and a control sequence.

Preferably, the frequency of occurrence of a sequence pattern in a testnucleic acid is compared with the frequency of occurrence in a sequenceknown to contain a STAR sequence. In this case a pattern is consideredrepresentative for a sequence comprising a STAR sequence if thefrequencies of occurrence are similar. Even more preferably, anothercriterion is used. The frequency of occurrence of a pattern in asequence comprising a STAR sequence is compared to the frequency ofoccurrence of the pattern in a control sequence. By comparing the twofrequencies it is possible to determine for each pattern thus analyzed,whether the frequency in the sequence comprising the STAR sequence issignificantly different from the frequency in the control sequence. Thena sequence pattern is considered to be representative of a sequencecomprising a STAR sequence, if the frequency of occurrence of thepattern in at least one sequence comprising a STAR sequence issignificantly different from the frequency of occurrence of the samepattern in a control sequence. By using larger numbers of sequencescomprising a STAR sequence the number of patterns for which astatistical difference can be established increases, thus enlarging thenumber of patterns for which the frequency of occurrence isrepresentative for a sequence comprising a STAR sequence. Preferably,the frequency of occurrence is representative of the frequency ofoccurrence of at least one sequence pattern in at least two sequencescomprising a STAR sequence, more preferably, in at least five sequencescomprising a STAR sequence. More preferably, in at least ten sequencescomprising a STAR sequence. More preferably, the frequency of occurrenceis representative of the frequency of occurrence of at least onesequence pattern in at least 20 sequences comprising a STAR sequence.Particularly preferred, the frequency of occurrence is representative ofthe frequency of occurrence of at least one sequence pattern in at least50 sequences comprising a STAR.

The patterns that are indicative for a sequence comprising a STARsequence are also dependent on the type of control nucleic acid used.The type of control sequence used is preferably selected on the basis ofthe sequence in which the presence of a STAR sequence is to be detected.Preferably, the control sequence comprises a random sequence comprisinga similar AT/CG content as at least one sequence comprising a STARsequence. Even more preferably, the control sequence is derived from thesame species as the sequence comprising the STAR sequence. For instance,if a test sequence is scrutinized for the presence of a STAR sequence,active in a plant cell, then preferably, the control sequence is alsoderived from a plant cell. Similarly, for testing for STAR activity in ahuman cell, the control nucleic acid is preferably also derived from ahuman genome. Preferably, the control sequence comprises between 50% and150% of the bases of at least one sequence comprising a STAR sequence.Particularly preferred, the control sequence comprises between 90% and110% of the bases of at least one sequence comprising a STAR sequence.More preferably, between 95% and 105%.

A pattern can comprise any number of bases larger than two. Preferably,at least one sequence pattern comprises at least five, more preferably,at least six bases. Even more preferably, at least one sequence patterncomprises at least eight bases. Preferably, at least one sequencepattern comprises a pattern listed in Table 4 and/or Table 5. A patternmay consist of a consecutive list of bases. However, the pattern mayalso comprise bases that are interrupted one or more times by a numberof bases that are not, or only, partly discriminative. A partlydiscriminative base is for instance indicated as a purine.

Preferably, the presence of STAR activity is verified using a functionalassay. Several methods are presented herein to determine whether asequence comprises STAR activity. STAR activity is confirmed if thesequence is capable of performing at least one of the followingfunctions: (i) at least in part inhibiting the effect of a sequencecomprising a gene transcription repressing element of the invention,(ii) at least in part blocking chromatin-associated repression, (iii) atleast in part blocking activity of an enhancer, (iv) conferring upon anoperably linked nucleic acid encoding a transcription unit compared tothe same nucleic acid alone. (iv-a) a higher predictability oftranscription, (iv-b) a higher transcription, and/or (iv-c) a higherstability of transcription over time.

The large number of sequences comprising STAR activity identified hereinopen up a wide variety of possibilities to generate and identifysequences comprising the same activity in kind, but not necessarily inamount. For instance, it is well within the reach of a skilled person toalter the sequences identified herein and test the altered sequence forSTAR activity. Such altered sequences are, therefore, also includedherein and can be used in a method for obtaining a cell which expressestwo or more proteins or in a method for identifying a cell whereinexpression of two or more proteins is in a predetermined ratio.Alteration can include deletion, insertion and mutation of one or morebases in the sequences.

Sequences comprising STAR activity were identified in stretches of 400bases. However, it is expected that not all of these 400 bases arerequired to retain STAR activity. Methods to delimit the sequences thatconfer a certain property to a fragment of between 400 and 5000 basesare well known. The minimal sequence length of a fragment comprisingSTAR activity is estimated to be about 50 bases.

Tables 4 and 5 list patterns of 6 bases that have been found to be overrepresented in nucleic acid molecules comprising STAR activity. Thisover representation is considered to be representative for a STARsequence. The tables were generated for a family of 65 STAR sequences.Similar tables can be generated starting from a different set of STARsequences, or from a smaller or larger set of STAR sequences. A patternis representative for a STAR sequence if it is over represented in theSTAR sequence compared to a sequence not comprising a STAR element. Thiscan be a random sequence. However, to exclude a non-relevant bias, thesequence comprising a STAR sequence is preferably compared to a genomeor a significant part thereof, more preferably, a genome of a vertebrateor plant, and even more preferably, a human genome. A significant partof a genome is for instance a chromosome. Preferably, the sequencecomprising a STAR sequence and the control sequence are derived from anucleic acid of the same species.

The more STAR sequences are used for the determination of the frequencyof occurrence of sequence patterns, the more representative for STARSthe patterns are that are over- or under-represented. Considering thatmany of the functional features that can be displayed by nucleic acidsare mediated by proteinaceous molecules binding to it, it is preferredthat the representative pattern is over-represented in the STARsequences. Such an over-represented pattern can be part of a bindingsite for such a proteinaceous molecule. Preferably, the frequency ofoccurrence is representative of the frequency of occurrence of at leastone sequence pattern in at least two sequences comprising a STARsequence, more preferably, in at least five sequences comprising a STARsequence, and even more preferably, in at least ten sequences comprisinga STAR sequence. More preferably, the frequency of occurrence isrepresentative of the frequency of occurrence of at least one sequencepattern in at least 20 sequences comprising a STAR sequence.Particularly preferred, the frequency of occurrence is representative ofthe frequency of occurrence of at least one sequence pattern in at least50 sequences comprising a STAR. Preferably, the sequences comprising aSTAR sequence comprise at least one of the sequences depicted in SEQ IDNOs:1-119.

STAR activity is a feature shared by the sequences listed in SEQ IDNOs:1-119. However, this does not mean that they must all share the sameidentifier sequence. It is very well possible that different identifiersexist. Identifiers may confer this common feature onto a fragmentcontaining it, though this is not necessarily so.

By using more sequences comprising STAR activity for determining thefrequency of occurrence of a sequence pattern or patterns, it ispossible to select patterns that are more often than others present orabsent in such a STAR sequence. In this way it is possible to findpatterns that are very frequently over or under represented in STARsequences. Frequently over or under represented patterns are more likelyto identify candidate STAR sequences in test sets. Another way of usinga set of over or under represented patterns is to determine whichpattern or combination of patterns is best suited to identify a STAR ina sequence. Using so-called discriminant statistics we have identified aset of patterns which performs best in identifying a sequence comprisinga STAR element. Preferably, at least one of the sequence patterns fordetecting a STAR sequence comprises a sequence pattern GGACCC (SEQ IDNO:505), CCCTGC (SEQ ID NO:247), AAGCCC (SEQ ID NO:311), CCCCCA (SEQ IDNO:339) and/or AGCACC (SEQ ID NO:377). Preferably, at least one of thesequence patterns for detecting a STAR sequence comprises a sequencepattern CCCN{16}AGC (SEQ ID NO:456), GGCN{9}GAC (SEQ ID NO:577),CACN{13}AGG (SEQ ID NO:802), and/or CTGN{4}GCC (SEQ ID NO:880).

A list of STAR sequences can also be used to determine one or moreconsensus sequences therein. A consensus sequence for a STAR element is,therefore, also provided herein. This consensus sequence can of coursebe used to identify candidate STAR elements in a test sequence.

Moreover, once a sequence comprising a STAR element has been identifiedin a vertebrate it can be used by means of sequence homology to identifysequences comprising a STAR element in other species belonging to avertebrate. Preferably, a mammalian STAR sequence is used to screen forSTAR sequences in other mammalian species. Similarly, once a STARsequence has been identified in a plant species it can be used to screenfor homologous sequences with similar function in other plant species.STAR sequences obtainable by a method as described herein are thusprovided. Further provided is a collection of STAR sequences.Preferably, the STAR sequence is a vertebrate or plant STAR sequence.More preferably, the STAR sequence is a mammalian STAR sequence or anangiosperm (monocot, such as rice or dicot, such as Arabidopsis). Morepreferably, the STAR sequence is a primate and/or human STAR sequence.

A list of sequences comprising STAR activity can be used to determinewhether a test sequence comprises a STAR element. There are, asmentioned above, many different methods for using such a list for thispurpose. Preferably, a method is provided for determining whether anucleic acid sequence of about 50-5000 base pairs comprises a STARsequence, this method comprising: generating a first table of sequencepatterns comprising the frequency of occurrence of the patterns in acollection of STAR sequences of the invention, generating a second tableof the patterns comprising the frequency of occurrence of the patternsin at least one reference sequence, selecting at least one pattern ofwhich the frequency of occurrence differs between the two tables,determining, within the nucleic acid sequence of about 50-5000 basepairs, the frequency of occurrence of at least one of the selectedpatterns, and determining whether the occurrence in the test nucleicacid is representative of the occurrence of the selected pattern in thecollection of STAR sequences. Alternatively, the determining stepcomprises determining whether the frequency of occurrence in the testnucleic acid is representative of the frequency of occurrence of theselected pattern in the collection of STAR sequences. Preferably, themethod further comprises determining whether the candidate STARcomprises a gene transcription modulating quality using a methoddescribed herein. Preferably, the collection of STARs comprises thesequences as depicted in SEQ ID NOs:1-119.

Now multiple methods are disclosed for obtaining a STAR sequence. Alsoprovided is an isolated and/or recombinant nucleic acid sequencecomprising a STAR sequence by a method as described herein.

A STAR sequence can exert its activity in a directional way, i.e., moreto one side of the fragment containing it than to the other. Moreover,STAR activity can be amplified in amount by multiplying the number ofSTAR elements. The latter suggests that a STAR element may comprise oneor more elements comprising STAR activity. Another way of identifying asequence capable of conferring STAR activity on a fragment containing itcomprises selecting from a vertebrate or plant sequence, a sequencecomprising STAR activity and identifying whether sequences flanking theselected sequence are conserved in another species. Such conservedflanking sequences are likely to be functional sequences. Such a methodfor identifying a sequence comprising a STAR element comprisingselecting a sequence of about 50 to 5000 base pairs from a vertebrate orplant species comprising a STAR element and identifying whethersequences flanking the selected sequence in the species are conserved inat least one other species. We further provide a method for detectingthe presence of a STAR sequence within a nucleic acid sequence of about50-5000 base pairs, comprising identifying a sequence comprising a STARsequence in a part of a chromosome of a cell of a species and detectingsignificant homology between the sequence and a sequence of a chromosomeof a different species. The STAR in the different species is thusidentified. Preferably, the species comprises a plant or vertebratespecies, preferably, a mammalian species. We also provide a method fordetecting the presence of a STAR element within a nucleic acid sequenceof about 50-5000 base pairs of a vertebrate or plant species, comprisingidentifying whether a flanking sequence of the nucleic acid sequence isconserved in at least one other species.

It is important to note that methods as disclosed herein for detectingthe presence of a sequence comprising a STAR sequence usingbioinformatical information are iterative in nature. The more sequencescomprising a STAR sequence that are identified with a method asdescribed herein, the more patterns are found to be discriminativebetween a sequence comprising a STAR sequence and a control sequence.Using these newly found discriminative patterns more sequencescomprising a STAR sequence can be identified which in turn enlarge theset of patterns that can discriminate, and so on. This iterative aspectis an important aspect of methods provided herein.

The present invention provides, amongst others, a method for obtaining acell which expresses two or more proteins, a method for identifying acell wherein expression of two or more proteins is in a predeterminedratio and a protein expression unit. The above-described obtainable STARsequences can be used. For example, a STAR sequence of SEQ ID NOs:1-119,Table 3, Table 4, or Table 5, or combinations thereof. More preferably,the STAR sequence is a vertebrate STAR sequence or a plant STARsequence. Even more preferably, the vertebrate STAR sequence is a humanSTAR sequence. It is furthermore preferred to use a STAR sequence from aspecies from which a gene of interest is expressed. For example, whenone would like to express two or more proteins and one of the proteinsis a human protein, one preferably includes a human STAR sequence forthe expression of the human protein.

As outlined above, the STAR elements flanking an expression unit are thebasis of the stable expression of the monomer transgenes over many cellgenerations. We have demonstrated that STAR elements can protectindividual transgenes from silencing. In the present invention thatcapability is extended to more than one expression unit introduced(preferentially) independently in a recombinant host cell. Expressionunits that are not flanked by STAR elements can undergo significantsilencing after only 5-10 culture passages, during which time silencingof the STAR protected units is negligible.

The advantages of a method for obtaining a cell which expresses two ormore proteins comprising providing the cell with two or more proteinexpression units encoding the two or more proteins, characterized inthat at least two of the protein expression units comprise at least oneSTAR sequence, are multifold.

The present invention uses STAR sequences for the production of two ormore proteins and thereby the invention provides (1) an increasedpredictability in the creation of recombinant cell lines thatefficiently produce the heterologous multimeric proteins of interest,(2) an increased yield of the heterologous multimeric proteins, (3)stable expression of the heterologous multimeric proteins, even duringprolonged cultivation in the absence of selection agent and (4) theinvention also provides favorable transgene expression characteristicswithout amplification of the transgene. The increased yield ofheterologous proteins provided by the invention may be obtained at lowtransgene copy numbers, without selective co-amplification using, forexample, the DHFR/methotrexate system. This results in greaterstability, since the transgene copy number is low and is not susceptibleto decrease due to recombination (McBurney et al., 2002) orrepeat-induced gene silencing (Garrick et al., 1998). Fifth, the broadapplicability of the method of the invention includes its utility in awide range of host cell lines. This is, for example, useful/desirablewhen a particular multimeric protein is preferably expressed by aparticular host cell line (e.g., expression of antibodies fromlymphocyte-derived host cell lines).

A method according to the invention, therefore, provides an improvementof expression of two or more proteins in a (host) cell.

In another embodiment, the invention provides a method for identifying acell wherein expression of two or more proteins is in a predeterminedratio comprising providing:

-   -   a collection of cells with two or more protein expression units        encoding the two or more proteins,    -   selecting cells which express two or more proteins, and    -   identifying from the obtained selection, cells that express two        or more proteins in the predetermined ratio, characterized in        that at least two of the protein expression units comprise at        least one STAR sequence.

The selection of cells which express two or more proteins may, forexample, be obtained by performing an SDS-PAGE analysis, a Western blotanalysis or an ELISA, which are all techniques which are known by aperson skilled in the art and, therefore, need no further elaboration.The identification of cells that express two or more proteins in thepredetermined ratio can also be performed by these techniques.

The presence of a STAR sequence in at least two of the proteinexpression units, again, provide the desired predictability, yield,stability and stoichiometrically balanced availability of the two ormore proteins.

Especially when polypeptides of a multimeric protein are producedaccording to a method of the invention it is desirable to provide therequired monomers/subunits in a ratio that is relevant for the formationof the multimeric protein. Hence, preferably, the monomers/subunits areproduced in a biological relevant balanced ratio. If, for example, amultimeric protein consists of two subunits A and one subunit B, it isdesired to produce two subunits A for every subunit of B that isproduced. Hence, a predetermined ratio is herein defined as the naturaloccurring ratio (stoichiometry) of the differentsubunits/monomers/polypeptides which comprise a multimeric protein.

In a more preferred embodiment, a cell obtainable according to a methodof the invention expresses two proteins. For example, two proteins whichtogether provide a therapeutically advantageous effect. In an even morepreferred embodiment, the predetermined ratio of the two expressedproteins is 1:1. This is, for example, useful in the production ofmultimeric proteins in which the monomers are in a 1:1 ratio. Typicalexamples are antibodies that comprise two heavy chains and two lightchains.

Preferably, the invention provides a method, wherein two or more proteinexpression units further encode at least two different selectionmarkers, and wherein the method further comprises a two-step selectionmarker screening on the cell, wherein the cell is selected in a firststep on the presence of a first selection marker and in a second step onthe presence of a second selection marker.

In this embodiment of the invention, a two-stage antibiotic selectionregime is used which results in a high proportion of isolates thatexpress, for example, transgenes 1 and 2 at high levels; the first stageof selection eliminates cells that do not contain the expression unit orunits, and the second stage of selection eliminates colonies that do nottranscribe both bicistronic mRNAs at high levels. This regime is one ofthe aspects for the increased frequency of multimer-expressingrecombinant cell lines achieved by the invention compared toconventional methods. As described herein, it results in an increase inthe frequency of expressor lines by more than ten-fold.

In another embodiment, the invention provides a method wherein at leastone of the protein expression units comprises a monocistronic genecomprising an open reading frame encoding a protein of interest andwherein the monocistronic gene is under control of a functionalpromoter.

In yet another embodiment, the invention provides a method, wherein atleast one of the protein expression units comprises: a bicistronic genecomprising an open reading frame encoding a protein of interest, aprotein translation initiation site with a reduced translationefficiency, and a selection marker, wherein the bicistronic gene isunder control of a functional promoter.

In a more preferred embodiment, the invention provides a method, whereinat least one of the protein expression units comprises: a bicistronicgene comprising an open reading frame encoding a protein of interest, aprotein translation initiation site with a reduced translationefficiency, and a selection marker, wherein the bicistronic gene isunder control of a functional promoter, which protein expression unitfurther comprises: a monocistronic gene comprising an open reading frameencoding a second selection marker, wherein the monocistronic gene isunder control of a functional promoter.

DESCRIPTION OF THE FIGURES

The drawings show representative versions of the DNA molecules of theinvention. These portions of DNA, referred to as (a) protein expressionunit(s), is/are created and manipulated in vectors such as recombinantplasmid molecules and/or recombinant viral genomes. The proteinexpression units are integrated into host cell genomes as part of themethod of the invention, and the schematic drawings represent theconfiguration of the DNA elements in the expression units in both thevector molecules and the host cell genome.

FIG. 1A is a schematic diagram that shows the first expression unit. Itis flanked by STAR elements, and comprises a bicistronic gene containing(from 5′ to 3′) a transgene (encoding, for example, a reporter gene orone subunit of a multimeric protein; TG S1, “transgene subunit 1”), anIRES, and a selectable marker (zeo, conferring zeocin resistance) undercontrol of the CMV promoter. A monocistronic selectable marker (neo,conferring G418 resistance) under control of the SV40 promoter isincluded. Both genes have the SV40 transcriptional terminator at their3′ ends (t).

FIG. 1B is a schematic diagram that shows the second expression unit. Itis flanked by STAR elements, and contains a bicistronic gene containing(from 5′ to 3′) a transgene (encoding, for example, a different reportergene or another subunit of a multimeric protein; TG S2), an IRES, and aselectable marker (bsd, conferring blasticidin resistance) under controlof the CMV promoter. A monocistronic selectable marker (neo, conferringG418 resistance) under control of the SV40 promoter is included. Bothgenes have the SV40 transcriptional terminator at their 3′ ends.

FIG. 2 is a diagram of the pSDH-CSP plasmid. The Secreted AlkalinePhosphatase (SEAP) reporter gene is under control of the CMV promoter,and the puromycin resistance selectable marker (puro) is under controlof the SV40 promoter. Flanking these two genes are multiple cloningsites into which STAR elements can be cloned. The plasmid also has anorigin of replication (ori) and ampicillin resistance gene (amp^(R)) forpropagation in Escherichia coli.

FIGS. 3A and 3B are diagrams of the pSDH-SIB/Z and pSDH-GIB/Z familiesof plasmids. These plasmids are derived from the pSDH-CSP plasmid (FIG.2), by replacement of the monocistronic SEAP and puro genes with abicistronic gene under control of the CMV promoter and a monocistronicneomycin resistance selectable marker gene (neo) under control of theSV40 promoter. FIG. 3A depicts pSDH-SIB/Z in which the bicistronic geneencodes secreted alkaline phosphatase (SEAP) in the 5′ position andblasticidin (bsd) or zeocin (zeo) resistance selectable markers in the3′ position, relative to the IRES. FIG. 3B illustrates pSDH-GIB/Z inwhich the bicistronic gene encodes green fluorescent protein (GFP) inthe 5′ position and blasticidin (bsd) or zeocin (zeo) resistanceselectable markers in the 3′ position, relative to the IRES.

FIG. 4 is a comparison of the consequences of one-step and two-stepantibiotic selection on the predictability of transgene expression.Recombinant CHO cell isolates containing plasmid pSDH-SIZ or plasmidpSDH-SIZ-STAR18 were selected on G418 (panel A) or sequentially on G418and zeocin (panel B) and assayed for SEAP activity.

FIG. 5 is a schematic diagram illustrating the PP (Plug and Play) familyof plasmids. These plasmids contain a bicistronic expression unit(containing an IRES) between multiple cloning sites (MCS) for insertionof STAR elements. MCSI, SbfI-SalI-XbaI-AscI-SwaI; MCSII,BsiWI-EcoRV-BglII-PacI. Panel A, the bicistronic gene encodes GFP andthe puromycin resistance marker (puro). Panel B, the bicistronic geneencodes secreted alkaline phosphatase (SEAP) and the zeocin resistancemarker (zeo). Panel C, the bicistronic gene encodes SEAP and the neocinresistance marker (neo). Panel D, the bicistronic gene encodes GFP andpuro, and an adjacent monocistronic gene encodes neo. Panel E, thebicistronic gene encodes SEAP and zeo, and an adjacent monocistronicgene encodes neo. Bicistronic genes are under control of the CMVpromoter (pCMV) and the monocistronic gene is under control of the SV40promoter (pSV40). A stuffer fragment of 0.37 kb (St) separates MCSI frompCMV. Both the bicistronic and monocistronic genes have the SV40polyadenylation site at their 3′ ends.

FIG. 6 is a diagram depicting the pSDH-CSP plasmid used for testing STARactivity. The Secreted Alkaline Phosphatase (SEAP) reporter gene isunder control of the CMV promoter, and the puromycin resistanceselectable marker (puro) is under control of the SV40 promoter. Flankingthese two genes are multiple cloning sites into which STAR elements canbe cloned. The plasmid also has an origin of replication (ori) andampicillin resistance gene (ampR) for propagation in Escherichia coli.

FIG. 7 is a graph illustrating that STAR6 (SEQ ID NO:6) and STAR49 (SEQID NO:49) improve predictability and yield of transgene expression.Expression of SEAP from the CMV promoter by CHO cells transfected withpSDH-CSP, pSDH-CSP-STAR6, or pSDH-CSP-STAR49 was determined. TheSTAR-containing constructs confer greater predictability and elevatedyield relative to the pSDH-CSP construct alone.

FIG. 8 is a graph illustrating that STAR6 (SEQ ID NO:6) and STAR8 (SEQID NO:8) improve predictability and yield of transgene expression.Expression of luciferase from the CMV promoter by U-2 OS cellstransfected with pSDH-CMV, pSDH-CMV-STAR6, or pSDH-CMV-STAR8 wasdetermined. The STAR-containing constructs confer greater predictabilityand elevated yield relative to the pSDH-CMV construct alone.

FIG. 9 is a graph showing the minimal essential sequences of STAR10 (SEQID NO:10) and STAR27 (SEQ ID NO:27). Portions of the STAR elements wereamplified by PCR: STAR10 (SEQ ID NO:10) was amplified with primers E23(SEQ ID NO:172) and E12 (SEQ ID NO:161) to yield fragment 10A, E13 (SEQID NO:162) and E14 (SEQ ID NO:163) to yield fragment 10B, and E15 (SEQID NO:164) and E16 (SEQ ID NO:165) to yield fragment 10C. STAR27 (SEQ IDNO:27) was amplified with primers E17 (SEQ ID NO:166) and E18 (SEQ IDNO:167) to yield fragment 27A, E19 (SEQ ID NO:168) and E20 (SEQ IDNO:169) to yield fragment 27B, and E21 (SEQ ID NO:170) and E22 (SEQ IDNO:171) to yield fragment 27C. These sub-fragments were cloned into thepSelect vector. After transfection into U-2 OS/Tet-Off/LexA-HP1 cells,the growth of the cultures in the presence of zeocin was monitored.Growth rates varied from vigorous (+++) to poor (+/−), while somecultures failed to survive zeocin treatment (−) due to absence of STARactivity in the DNA fragment tested.

FIG. 10 is a graph showing STAR element function in the context of theSV40 promoter. pSDH-SV40 and pSDH-SV40-STAR6 were transfected into thehuman osteosarcoma U-2 OS cell line, and expression of luciferase wasassayed with or without protection from gene silencing by STAR6 (SEQ IDNO:6) in puromycin-resistant clones.

FIG. 11 is a graph illustrating STAR element function in the context ofthe Tet-Off promoter. pSDH-Tet and pSDH-Tet-STAR6 were transfected intothe human osteosarcoma U-2 OS cell line, and expression of luciferasewas assayed with or without protection from gene silencing by STAR6 (SEQID NO:6) in puromycin-resistant clones.

FIG. 12 is a schematic diagram illustrating the orientation of STARelements as they are cloned in the pSelect vector (panel A), as they arecloned into pSDH vectors to preserve their native orientation (panel B),and as they are cloned into pSDH vector in the opposite orientation(panel C).

FIG. 13 is a graph showing directionality of STAR66 (SEQ ID NO:66)function. The STAR66 element (SEQ ID NO:66) was cloned into pSDH-Tet ineither the native (STAR66 native) or the opposite orientation (STAR66opposite), and transfected into U-2 OS cells. Luciferase activity wasassayed in puromycin resistant clones.

FIG. 14 is a southern blot showing copy number-dependence of STARfunction. Southern blot of luciferase expression units inpSDH-Tet-STAR10, integrated into U-2 OS genomic DNA. Radioactiveluciferase DNA probe was used to detect the amount of transgene DNA inthe genome of each clone, which was then quantified with aphosphorimager.

FIG. 15 is a graph illustrating copy number-dependence of STAR function.The copy number of pSDH-Tet-STAR10 expression units in each clone wasdetermined by phosphorimagery, and compared with the activity of theluciferase reporter enzyme expressed by each clone.

FIG. 16 contains graphs illustrating enhancer-blocking and enhancerassays. The luciferase expression vectors used for testing STARs forenhancer-blocking and enhancer activity are shown schematically. TheE-box binding site for the E47 enhancer protein is upstream of a cloningsite for STAR elements. Downstream of the STAR cloning site is theluciferase gene under control of a human alkaline phosphatase minimalpromoter (mp). The histograms indicate the expected outcomes for thethree possible experimental situations (see text). Panel A:Enhancer-blocking assay. Panel B: Enhancer assay.

FIG. 17 is a graph depicting the enhancer-blocking assay. Luciferaseexpression from a minimal promoter is activated by the E47/E-boxenhancer in the empty vector (vector). Insertion of enhancer-blockers(scs, HS4) or STAR elements (STAR elements 1, 2, 3, 6, 10, 11, 18, and27; SEQ ID NOS: 1, 2, 3, 6, 10, 11, 18, and 27, respectively) blockluciferase activation by the E47/E-box enhancer.

FIG. 18 is a graph illustrating an enhancer assay. Luciferase expressionfrom a minimal promoter is activated by the E47/E-box enhancer in theempty vector (E47). Insertion of the scs and HS4 elements or variousSTAR elements (STARs 1, 2, 3, 6, 10, 11, 18, and 27; SEQ ID NOS: 1, 2,3, 6, 10, 11, 18, and 27, respectively) do not activate transcription ofthe reporter gene.

FIG. 19 contains graphs illustrating STAR18 (SEQ ID NO:18) sequenceconservation between mouse and human. The region of the human genomecontaining 497 base pair STAR18 (SEQ ID NO:18) is shown (black boxes);the element occurs between the HOXD8 and HOXD4 homeobox genes on humanchromosome 2. It is aligned with a region in mouse chromosome 2 thatshares 72% sequence identity. The region of human chromosome 2immediately to the left of STAR18 is also highly conserved with mousechromosome 2 (73% identity; gray boxes); beyond these region, theidentity drops below 60%. The ability of these regions from human andmouse, either separately or in combination, to confer growth on zeocinis indicated: −, no growth; +, moderate growth; ++, vigorous growth;+++, rapid growth.

FIG. 20 is a schematic diagram of bio-informatical analysis workflow.For details, see text.

FIG. 21 is a diagram illustrating the results of discriminant analysison classification of the training set of 65 STAR elements. STAR elementsthat are correctly classified as STARs by Stepwise Linear DiscriminantAnalysis (LDA) are shown in a Venn diagram. The variables for LDA wereselected from frequency analysis results for hexameric oligonucleotides(“oligos”) and for dyads. The diagram indicates the concordance of thetwo sets of variables in correctly classifying STARs.

FIG. 22 is a graph depicting the RT-PCR assay of Arabidopsis STARstrength. U-2 OS/Tet-Off/lexA-HP1 cells were transfected with candidateArabidopsis STAR elements and cultivated at low doxycyclineconcentrations. Total RNA was isolated and subjected to RT-PCR; thebands corresponding to the zeocin and hygromycin resistance mRNAs weredetected by Southern blotting and quantified with a phosphorimager. Theratio of the zeocin to hygromycin signals is shown for transfectantscontaining zeocin expression units flanked by 12 different ArabidopsisSTAR elements, the Drosophila scs element, or no flanking element.

FIG. 23 includes schematic diagrams and a graph illustrating that STARelements allow efficient and simultaneous expression of two genes fromtwo distant vectors. The ppGIZ, ppGIZ-STAR7, ppRIP and ppRIP-STAR7vectors used for testing simultaneous expression of respectively GFP andRED are shown. The expression unit comprises (from 5′ to 3′) genesencoding the GFP or RED proteins, an IRES, and a selectable marker (zeo,conferring zeocin resistance or respectively puro, puromycin resistancegene) under control of the CMV promoter. The expression unit has theSV40 transcriptional terminator at its 3′ end (t). The cassettes withthe GFP and RED expression units are either flanked by STAR7 elements(SEQ ID NO:7) (STAR7-shielded) or not (Control). The two controlconstructs or the two STAR7-shielded vectors are simultaneouslytransfected to CHO-K1 cells. Stable colonies that are resistant to bothzeocin and puromycin are expanded and the GFP and RED signals aredetermined on a XL-MCL Beckman Coulter flow cytometer. The percentage ofcells in one colony that are double positive for both GFP and REDsignals is taken as measure for simultaneous expression of both proteinsand this is plotted in FIG. 23.

FIG. 24 also includes schematic diagrams and a graph illustrating thatSTAR elements improve expression of a functional antibody in CHO cells.The different vectors containing the Light and Heavy Chain of the RING1antibody are shown in FIG. 24. The constructs are simultaneouslytransfected to CHO cells. Stable colonies that are resistant to bothzeocin and puromycin are expanded. The cell culture medium of thesecolonies is tested for the detection of functional RING1 antibody in anELISA with RING1 protein as antigen. The values are dividing by thenumber of cells in the colony. The highest value detected in theSTAR-less control is arbitrarily set at 100%.

DETAILED DESCRIPTION OF THE INVENTION Definitions Cell, Host Cell, CellLine, Host Cell Line

The terms “cell”/“host cell” and “cell line”/“host cell line” arerespectively typically defined as a eukaryotic cell and homogeneouspopulations thereof that are maintained in cell culture by methods knownin the art, and that have the ability to express heterologous proteins.

Expression

The term “expression” is typically used to refer to the production of aspecific RNA product or products, or a specific protein or proteins, ina cell. In the case of RNA products, it refers to the process oftranscription. In the case of protein products, it refers to theprocesses of transcription, translation and optionallypost-translational modifications. In the case of secreted proteins, itrefers to the processes of transcription, translation, and optionallypost-translational modification (e.g., glycosylation, disfulfide bondformation, etc.), followed by secretion. In the case of multimericproteins, it includes assembly of the multimeric structure from thepolypeptide monomers. The corresponding verbs of the noun “expression”have an analogous meaning as the noun.

Protein, Multimer, Multimeric Protein

A protein is herein defined as being either (i) a product obtained bythe processes of transcription and translation and possibly but notnecessarily a product that is part of a multimeric protein (for example,a subunit) and/or (ii) a product obtained by the processes oftranscription, translation and post-translational modification. The term“multimer” or “multimeric protein” is typically defined as a proteinthat comprises two or more, possibly non-identical, polypeptide chains(“monomers”). The different monomers in a multimeric protein can bepresent in stoichiometrically equal or unequal numbers. In either case,the proportion of the monomers is usually fixed by the functionalstructure of the multimeric protein.

Protein Expression Unit

The term “protein expression unit” is herein defined as a unit capableof providing protein expression and typically comprises a functionalpromoter, an open reading frame encoding a protein of interest and afunctional terminator, all in operable configuration. A functionalpromoter is a promoter that is capable of initiating transcription in aparticular cell. Suitable promoters for obtaining expression ineukaryotic cells are the CMV-promoter, a mammalian EF1-alpha promoter, amammalian ubiquitin promoter, or a SV40 promoter. A functionalterminator is a terminator that is capable of providing transcriptiontermination. One example of a suitable terminator is an SV40 terminator.

An Open Reading Frame Encoding a Protein of Interest (or a Transgene)

The term “an open reading frame encoding a protein of interest (or atransgene)” is typically defined as a fragment of DNA which codes for aspecific RNA product or products or a specific protein or proteins, andwhich is optionally capable of becoming integrated into the genome of ahost cell. It includes DNA elements required for proper transcriptionand translation of the coding region(s) of the transgene. The DNAencoding the protein of interest/transgene can either be a DNA encodinga product obtained by the processes of transcription and translation(and possibly but not necessarily this product is part of a multimericprotein, for example, a subunit) or a product obtained by the processesof transcription, translation and post-translational modification.

Recombinant Cell, Recombinant Host Cell, Recombinant Cell Line,Recombinant Host Cell Line

The terms “recombinant cell/host cell” and “recombinant cell line/hostcell line” are respectively typically defined as a host cell andhomogeneous populations thereof into which a transgene has beenintroduced for the purpose of producing a heterologous protein orproteins.

STAR (Stabilizing Anti-Repressor) Sequence & STAR Element

A STAR (STabilizing Anti-Repressor) sequence (or STAR element; theseterms will be used interchangeably herein) is a naturally occurring DNAelement that we have isolated from eukaryotic genomes on the basis oftheir ability to block transgene repression. Preferably, the STARelements are recovered from the human genome. A STAR sequence comprisesthe capacity to influence transcription of genes in cis and/or provide astabilizing and/or an enhancing effect. It has been demonstrated thatwhen STAR elements flank transgenes, the transgene expression level ofrandomly selected recombinant cell lines can be increased to levelsapproaching the maximum potential expression of the transgene'spromoter. Moreover, the expression level of the transgene is stable overmany cell generations, and does not manifest stochastic silencing.Therefore, STAR sequences confer a degree of position-independentexpression on transgenes that is not possible with conventionaltransgenic systems. The position independence means that transgenes thatare integrated in genomic locations that would result in transgenesilencing are, with the protection of STAR elements, maintained in atranscriptionally active state.

Quality

The term quality in relation to a sequence refers to an activity of thesequence.

STAR, STAR Sequence, STAR Element

The term STAR, STAR sequence or STAR element, as used herein, refers toa DNA sequence comprising one or more of the mentioned genetranscription modulating qualities.

DNA Sequence

The term “DNA sequence” as used herein does, unless otherwise specified,not refer to a listing of specific ordering of bases but rather to aphysical piece of DNA. A transcription quality with reference to a DNAsequence refers to an effect that the DNA sequence has on transcriptionof a gene of interest. “Quality” as used herein refers to detectableproperties or attributes of a nucleic acid or protein in a transcriptionsystem.

Bicistronic Gene

The term “bicistronic gene,” is typically defined as a gene capable ofproviding a RNA molecule that encodes two proteins/polypeptides.

Monocistronic Gene

The term “monocistronic gene” is typically defined as a gene capable ofproviding a RNA molecule that encodes one protein/polypeptide.

Selection Marker or Selectable Marker

The term “selection marker or selectable marker” is typically used torefer to a gene and/or protein whose presence can be detected directlyor indirectly in a cell, for example, a gene and/or a protein thatinactivates a selection agent and protects the host cell from theagent's lethal or growth-inhibitory effects (e.g., an antibioticresistance gene and/or protein). Another possibility is that theselection marker induces fluorescence or a color deposit (e.g., greenfluorescent protein and derivatives, luciferase, or alkalinephosphatase).

Selection Agent

The term “selection agent” is typically defined as a chemical compoundthat is able to kill or retard the growth of host cells (e.g., anantibiotic).

Selection

The term “selection” is typically defined as the process of using aselection marker/selectable marker and a selection agent to identifyhost cells with specific genetic properties (e.g., that the host cellcontains a transgene integrated into its genome).

Clone, Isolate

The nouns “clone” and “isolate” typically refer to a recombinant hostcell line that has been identified and isolated by means of selection.

The improvements provided by a method according to the invention havethree integrated aspects. (1) With existing systems, recombinant celllines that simultaneously express acceptable quantities of the monomersof multimeric proteins can be created only at very low frequencies; thepresent invention increases the predictability of creating high-yieldingrecombinant host cell lines by a factor of ten or more. (2) Existingsystems do not provide stoichiometrically balanced and proportionalamounts of the subunits of multimeric proteins; the present inventionensures that the expression levels of the subunits will be balanced andproportional. (3) Existing systems do not provide a means of protectingthe transgenes that encode the protein subunits from transgenesilencing.

FIG. 1 provides a non-limiting schematic representation of one of theembodiments of this part of the invention. FIGS. 1A and 1B show twoseparate protein expression units. This is the configuration of the DNAelements of the expression units in the plasmid as well as afterintegration into the genome. Expression unit one is shown in FIG. 1A. Itcontains an open reading frame for a transgene (a reporter gene orsubunit 1 of a multimeric (TG S1, transgene subunit 1)). This isupstream of the attenuated EMCV IRES, and of the open reading frameencoding the zeocin resistance selectable marker protein (zeo). Thisbicistronic transgene is transcribed at high levels from the CMVpromoter. Next to this is the neomycin resistance selectable marker(neo; this confers resistance to the antibiotic G418 as well),transcribed as a monocistronic mRNA from the SV40 promoter. These twogenes are flanked by STAR elements. In FIG. 1B a similar expression unitis depicted. It consists of a second transgene (a second reporter geneor the open reading frame for subunit 2 of a heterodimeric protein (TGS2)) upstream of the attenuated EMCV IRES and the blasticidin selectablemarker open reading frame (bsd). This bicistronic transgene istranscribed at high levels from the CMV promoter. Next to this is theneo selectable marker, transcribed as a monocistronic mRNA from the SV40promoter. The two genes in the second expression unit are flanked bySTAR elements as well.

The possible combinations of selection markers are numerous. Examples ofpossible antibiotic combinations are provided herein. The one antibioticthat is particularly advantageous is zeocin, because thezeocin-resistance protein (zeocin-R) acts by binding the drug andrendering it harmless. Therefore, it is easy to titrate the amount ofdrug that kills cells with low levels of zeocin-R expression, whileallowing the high-expressors to survive. All other antibiotic-resistanceproteins in common use are enzymes, and thus act catalytically (not 1:1with the drug).

When a two-step selection is performed it is, therefore, advantageous touse an antibiotic resistance protein with this 1:1 binding mode ofaction. Hence, the antibiotic zeocin is a preferred selection marker.For convenience the zeocin antibiotic is in a two-step selection methodcombined with puromycin-R or blasticidin-R in the second bicistronicgene, and neomycin-R or hygromycin-R in the monocistronic gene.

It is also possible to combine an antibiotic selection marker with aselection marker which provides induction of fluorescence or whichprovides a color deposit.

Different promoters can be used as long as they are functional in theused cell. The CMV promoter is considered the strongest available, so itis preferably chosen for the bicistronic gene in order to obtain thehighest possible product yield. Other examples of suitable promotersare, e.g., mammalian promoters for EF1-alpha or ubiquitin. The goodexpression and stability of the SV40 promoter makes it well suited forexpression of the monocistronic gene; enough selection marker protein(for example, the antibiotic resistance protein neomycin-R in theexample cited herein) is made to confer high expression of the selectionmarker. Hence, the SV40 promoter is preferentially used as a promoterdriving the expression of the selection marker.

In a preferred embodiment, the invention provides a method wherein atleast one of the protein expression units comprises at least two STARsequences. In an even more preferred embodiment, the invention providesa method wherein the protein expression unit comprising at least twoSTAR sequences is arranged such that the protein expression unit isflanked on either side by at least one STAR sequence. In yet an evenmore preferred embodiment, the at least two STAR sequences areessentially identical. Essentially identical STAR sequences are definedherein as STAR sequences which are identical in their important domains,but which may vary within their less important domains (the domains thatconfer the transcription stabilizing or enhancing quality), for example,a point mutation, deletion or insertion at a less important positionwithin the STAR sequence. Preferentially, the essentially identical STARsequences provide equal amounts of transcription stabilizing orenhancing activity.

The use of STAR sequences to flank at least one protein expression unitis one of the aspects of the balanced and proportional levels ofexpression of two or more proteins and more specifically for theexpression of the monomers of multimeric proteins. The STAR sequencescreate chromatin domains of definite and stable transcriptionalpotential. As a result, promoters that drive transcription of eachbicistronic mRNA will function at definite, stable levels. A recombinanthost cell line created by the method of the invention is readilyidentified in which these levels result in appropriate proportions ofeach monomer of the multimeric protein of interest being expressed athigh yields.

In another embodiment, the protein expression unit contains only thebicistronic gene flanked by STAR elements. The advantages of omittingthe monocistronic antibiotic resistance gene are twofold. First,selection of high-expressing recombinant host cells requires the use ofonly two antibiotics. Second, it prevents repression of the bicistronicand/or monocistronic genes by the phenomena of promoter suppression andtranscriptional interference. These phenomena are common problems inconventional transgenic systems in which two or more transcription unitsare located near each other. Repression by an upstream (5′) unit of adownstream (3′) unit is termed transcriptional interference, andrepression by a downstream unit of an upstream unit is termed promotersuppression (Villemure et al., 2001). Transcriptional interference canresult in suppression of adjacent transgenes in all possiblearrangements (tandem, divergent, and convergent) (Eszterhas et al.,2002). These phenomena can reduce the efficiency of selection of theIRES-dependent and/or monocistronic antibiotic resistance genes, andreduce the yield of the transgene. Therefore, the embodiment of theinvention comprising only a bicistronic gene flanked by STAR elementsprovides an alternative configuration of the components.

In a preferred embodiment, the method according to the invention uses aSTAR sequence wherein the STAR sequence is depicted in Table 3 and/orSEQ ID NOs:1-119 and/or a functional equivalent and/or a functionalfragment thereof.

We have isolated and characterized an extensive collection of STARsequences using proprietary technology. The strength of these sequencesranges widely. This is manifested by the varying degrees of improvementof transgene expression in recombinant host cells conferred by the STARelements; some STAR elements provide full protection from silencing,while others only provide partial protection. The range in strength ofthe STAR elements is also manifested in their varying capacities toimprove the predictability of isolating recombinant cell lines thatefficiently produce the heterologous proteins of interest. For thepresent invention we have preferably employed STAR elements that havestrong predictability characteristics, in order to have high numbers ofefficiently-expressing recombinant cell lines. The STAR elementsemployed have moderate to strong anti-repressor activity, in order to beable to modulate the levels of recombinant protein production to matchthe requirements of the product (e.g., balanced and proportionalexpression of polypeptide monomers). The selected STAR elements alsoconfer significant increases on the stability of expression of thetransgenes.

Some STAR elements also display promoter and host cell-type specificity.These characteristics are exploited to create novel transgenic systemsto optimize the production of heterologous proteins that require aspecific host cell (for example, to achieve a high yield or apharmaceutically advantageous glycosylation pattern) or a specific modeof expression (for example, the use of an inducible promoter or aconstitutive promoter; the use of a promoter with moderate strength orhigh strength, etc.). Therefore, the use of different STAR elementsresults in different embodiments of the invention that pertain to thesetypes of applications.

A functional equivalent and/or a functional fragment of a sequencedepicted in Table 3 and/or SEQ ID NOs:1-119 is defined herein asfollows. A functional equivalent of a sequence as depicted in Table 3and/or SEQ ID NOs:1-119 is a sequence derived with the information givenin Table 3 and/or SEQ ID NOs:1-119. For instance, a sequence that can bederived from a sequence in Table 3 and/or SEQ ID NOs:1-119 by deleting,modifying and/or inserting bases in or from a sequence listed in Table 3and/or SEQ ID NOs:1-119, wherein the derived sequence comprises the sameactivity in kind, not necessarily in amount, of a sequence as depictedin Table 3 and/or SEQ ID NOs:1-119. A functional equivalent is further asequence comprising a part from two or more sequences depicted in Table3 and/or SEQ ID NOs:1-119. A functional equivalent can also be asynthetic DNA sequence which is a sequence that is not derived directlyor indirectly from a sequence present in an organism. For instance, asequence comprising a drosophila scs or scs′ sequence is not a syntheticsequence, even when the scs or scs′ sequence was artificially generated.

Functional sequences of STAR elements can be delineated by variousmethods known in the art. In one embodiment, deletions and/orsubstitutions are made in STAR sequences. DNA that is modified in such away is, for example, tested for activity by using a single modifiednucleic acid or by generating a collection of test nucleic acidscomprising the modified nucleic acid. Elucidation of functionalsequences within STAR sequences enables the elucidation of consensussequences for elements with a gene transcription modulating and/or agene transcription repressing quality.

A functional fragment of a STAR sequence as depicted in Table 3 and/orSEQ ID NOs:1-119 can, for example, be obtained by deletions from the 5′end or the 3′ end or from the inside of the sequences or any combinationthereof, wherein the derived sequence comprises the same activity inkind, not necessarily in amount.

In a more preferred embodiment, the STAR sequence as depicted in Table 3and/or SEQ ID NOs:1-119 is STAR18 and/or a functional equivalent and/ora functional fragment thereof.

Yet another preferred feature of a method according to the invention isthe introduction of a (weak) IRES as an example of a protein translationinitiation site with a reduced translation efficiency, between the openreading frame of the protein of interest and the selection marker openreading frame. In combination with, for example, the STAR sequence, thiscomponent of the present invention comprises a marked improvement intransgenic systems for the expression of two or more proteins.

IRES elements are known from viral and mammalian genes (Martinez-Salas,1999), and have also been identified in screens of small syntheticoligonucleotides (Venkatesan & Dasgupta, 2001). The IRES from theencephalomyocarditis virus has been analyzed in detail (Mizuguchi etal., 2000). An IRES is an element encoded in DNA that results in astructure in the transcribed RNA at which eukaryotic ribosomes can bindand initiate translation. An IRES permits two or more proteins to beproduced from a single RNA molecule (the first protein is translated byribosomes that bind the RNA at the cap structure of its 5′ terminus,(Martinez-Salas, 1999)). Translation of proteins from IRES elements isless efficient than cap-dependent translation: the amount of proteinfrom IRES-dependent open reading frames (ORFs) ranges from less than 20%to 50% of the amount from the first ORF (Mizuguchi et al., 2000). Thisrenders IRES elements undesirable for production of all subunits of amultimeric protein from one messenger RNA (mRNA), since it is notpossible to achieve balanced and proportional expression of two or moreprotein monomers from a bicistronic or multicistronic mRNA. However, thereduced efficiency of IRES-dependent translation provides an advantagethat is exploited by the current invention. Furthermore, mutation ofIRES elements can attenuate their activity, and lower the expressionfrom the IRES-dependent ORFs to below 10% of the first ORF (Lopez deQuinto & Martinez-Salas, 1998, Rees et al., 1996). The advantageexploited by the invention is as follows: when the IRES-dependent ORFencodes a selectable marker protein, its low relative level oftranslation means that high absolute levels of transcription must occurin order for the recombinant host cell to be selected. Therefore,selected recombinant host cell isolates will by necessity express highamounts of the transgene mRNA. Since the recombinant protein istranslated from the cap-dependent ORF, it can be produced in abundanceresulting in high product yields.

Changes to the IRES can be made without altering the essence of thefunction of the IRES (hence, providing a protein translation initiationsite with a reduced translation efficiency), resulting in a modifiedIRES. Use of a modified IRES which is still capable of providing a smallpercentage of translation (compared to a 5′ cap translation) is,therefore, also included in this invention.

In yet another embodiment, the invention provides a method for obtaininga cell which expresses two or more proteins or a method for identifyinga cell wherein expression of two or more proteins is in a predeterminedratio, wherein each of the protein expression units resides on aseparate DNA-carrier. The present invention preferentially makes use ofa separate transcription unit for each protein and/or monomer of amultimeric protein. In each transcription unit the monomer ORF isproduced by efficient cap-dependent translation. This feature of theinvention provides isolated recombinant host cells that have high yieldsof each monomer, at levels that are balanced and proportionate to thestoichiometry of the multimeric protein. The increased predictability atwhich such recombinant host cells are isolated results in an improvementin the efficiency of screening for such isolates by a factor of ten ormore. In a preferred embodiment, the DNA-carrier is a vector (orplasmid; the terms are used interchangeably herein). In anotherembodiment, the vector is a viral vector and in a more preferredembodiment, the viral vector is an adenoviral vector or a retroviralvector. Other viral vectors can also be used in a method according tothe invention.

Conventional expression systems are DNA molecules in the form of arecombinant plasmid or a recombinant viral genome. The plasmid or theviral genome is introduced into (mammalian host) cells and integratedinto their genomes by methods known in the art. The present inventionalso uses these types of DNA molecules to deliver its improved transgeneexpression system. A preferred embodiment of the invention is the use ofplasmid DNA for delivery of the expression system. A plasmid contains anumber of components: conventional components, known in the art, are anorigin of replication and a selectable marker for propagation of theplasmid in bacterial cells; a selectable marker that functions ineukaryotic cells to identify and isolate host cells that carry anintegrated transgene expression system; the protein of interest, whosehigh-level transcription is brought about by a promoter that isfunctional in eukaryotic cells (e.g., the human cytomegalovirus majorimmediate early promoter/enhancer, pCMV (Boshart et al., 1985)); andviral transcriptional terminators for the transgene of interest and theselectable marker (e.g., the SV40 polyadenylation site (Kaufman & Sharp,1982)).

The vector used can be any vector that is suitable for cloning DNA andthat can be used in a transcription system. When host cells are used itis preferred that the vector is an episomally replicating vector. Inthis way, effects due to different sites of integration of the vectorare avoided. DNA elements flanking the vector at the site of integrationcan have effects on the level of transcription of the promoter andthereby mimic effects of fragments comprising DNA sequences with a genetranscription modulating quality. In a preferred embodiment, the vectorcomprises a replication origin from the Epstein-Barr virus (EBV), OriP,and a nuclear antigen (EBNA-1). Such vectors are capable of replicatingin many types of eukaryotic cells and assemble into chromatin underappropriate conditions.

In a preferred embodiment, the invention provides a method for obtaininga cell which expresses two or more proteins or a method for obtaining acell wherein expression of two or more proteins is in a predeterminedratio comprising providing two or more protein expression units whereinone of the protein expression units or protein(s) of interest encodes animmunoglobulin heavy chain and/or wherein another of the proteinexpression units or protein(s) of interest encodes an immunoglobulinlight chain. According to this embodiment, a multimeric protein, anantibody, is obtained. It is possible to provide a cell which expressesan immunoglobulin heavy chain from one protein expression unit and animmunoglobulin light chain from another protein expression unit with athird protein expression unit encoding a secretory component or ajoining chain. In this way the production of, for example, sIgA andpentameric IgM is provided.

Preferably, the used host cell secretes the produced multimer. In thisway the product is easily isolated from the medium surrounding the hostcell.

More preferably, the invention results in the production of a functionalmultimer. The functionality of the produced multimer is determined withstandard procedures. For example, a produced multi subunit enzyme istested in a corresponding enzymatic assay or by binding to an antigen,for example, in an ELISA used to test the functionality of a producedantibody.

Hence, the selection of a final suitable host cell expressing a multimerinvolves multiple steps amongst which are the selection for a cell thatexpresses all the desired subunits of a multimer, followed by afunctional analysis of the multimer.

With regard to a multimeric protein, high expression levels of thesubunits is desired as well as the formation of a functional multimericprotein of the subunits. Surprisingly, the use of a STAR sequence forthe production of the subunits of a multimeric protein results in a highamount of cells that express the subunits, as compared to controlvectors without a STAR sequence. Moreover, the amount of functionalmultimeric protein is relatively higher when compared to the control.

Production of subunits and the formation of functional multimericprotein from these subunits is in particular of importance for theproduction of antibodies. When the heavy chain and light chainexpression cassette are flanked by a STAR sequence this results in ahigher production of functional antibody, as compared to control vectorswithout a STAR sequence. Hence, the presence of a STAR sequence resultsin a higher degree of predictability of functional antibody expression.Preferably, each expression unit comprises at least two STAR sequenceswhich sequences are arranged such that the expression unit is flanked oneither side by at least one STAR sequence.

In yet another embodiment, a method according to the invention isprovided, wherein the protein expression units are introducedsimultaneously into the cell.

Preferably, a functional promoter is a human cytomegalovirus (CMV)promoter, a simian virus (SV40) promoter, a human ubiquitin C promoteror a human elongation factor alpha (EF1-α) promoter.

As disclosed herein within the experimental part, a STAR sequence canconfer copy number-dependence on a transgene expression unit, makingtransgene expression independent of other transgene copies in tandemarrays, and independent of gene-silencing influences at the site ofintegration. Hence, the invention also provides a method for obtaining acell which expresses two or more proteins or a method for identifying acell wherein expression of two or more proteins is in a predeterminedratio in which multiple copies of a protein expression unit encoding aprotein of interest is integrated into the genome of the cell (i.e., inwhich cell, an amplification of the gene of interest is present).

According to this part of the invention, the protein expression unitsare introduced simultaneously into the (host) cell or collection ofcells by methods known in the art. Recombinant host cells are selectedby treatment with an appropriate antibiotic, for example, G418, usingmethods known in the art. After formation of individualantibiotic-resistant colonies, another antibiotic or a combination ofantibiotics, for example, a combination of zeocin and blasticidin,is/are applied, and antibiotic-resistant colonies are identified andisolated. These are tested for the level of expression of transgenes.

In another embodiment, the invention provides a protein expression unitcomprising:

-   -   a bicistronic gene comprising an open reading frame encoding a        protein of interest, a protein translation initiation site with        a reduced translation efficiency, a selection marker and wherein        the bicistronic gene is under control of a functional promoter    -   at least one STAR sequence.

In a more preferred embodiment, the protein expression unit furthercomprises: a monocistronic gene comprising an open reading frameencoding a second selection marker and wherein the monocistronic gene isunder control of a functional promoter.

In an even more preferred embodiment, the protein expression unitcomprises at least two STAR sequences which are preferentially arrangedsuch that the protein expression unit is flanked on either side by atleast one STAR sequence. Examples of such a protein expression unit areprovided within the experimental part of this patent application (forexample, FIGS. 1 and 5).

In another embodiment, the protein expression unit according to theinvention comprises STAR sequences, wherein the STAR sequences areessentially identical.

In a preferred embodiment, the invention provides a protein expressionunit comprising:

-   -   a bicistronic gene comprising an open reading frame encoding a        protein of interest, a protein translation initiation site with        a reduced translation efficiency, a selection marker and wherein        the bicistronic gene is under control of a functional promoter    -   at least one STAR sequence, and is optionally provided with a        monocistronic gene cassette, wherein the STAR sequence is        depicted in Table 3 and/or SEQ ID NOs:1-119 and/or a functional        equivalent and/or a functional fragment thereof and even more        preferred wherein the STAR sequence is STAR18.

In another embodiment, a protein expression unit according to theinvention is provided wherein the protein translation initiation sitewith a reduced translation efficiency comprises an IRES. Morepreferably, a modified, e.g., weaker, IRES is used.

In yet another embodiment, a protein expression unit according to theinvention is provided wherein the protein expression unit is a vector.In a preferred embodiment, the DNA-carrier is a vector (or plasmid; theterms are used interchangeably herein). In another embodiment, thevector is a viral vector and in a more preferred embodiment, the viralvector is an adenoviral vector or a retroviral vector. Other viralvectors can also be used in a method according to the invention.

In a preferred embodiment, a protein expression unit according to theinvention is provided, wherein the protein of interest is animmunoglobulin heavy chain. In yet another preferred embodiment, aprotein expression unit according to the invention is provided, whereinthe protein of interest is an immunoglobulin light chain. When these twoprotein expression units are present within the same (host) cell amultimeric protein and more specifically an antibody is assembled.

The invention includes a cell provided with a protein expression unitcomprising a STAR.

The invention also includes a (host) cell comprising at least oneprotein expression unit according to the invention. Such a (host) cellis then, for example, used for large-scale production processes.

The invention also includes a cell obtainable according to anyone of themethods as described herein. The invention furthermore includes aprotein obtainable from the cell (for example, via the process ofprotein purification). Preferably, the protein is a multimeric proteinand even more preferably, the multimeric protein is an antibody. Such anantibody can be used in pharmaceutical and/or diagnostic applications.

The foregoing discussion and the following examples are provided forillustrative purposes, and they are not intended to limit the scope ofthe invention as claimed herein. They simply provide some of thepreferred embodiments of the invention. Modifications and variations,which may occur to one of ordinary skill in the art, are within theintended scope of this invention. Various other embodiments apply to thepresent invention, including: other selectable marker genes; other IRESelements or means of attenuating IRES activity; other elements affectingtranscription including promoters, enhancers, introns, terminators, andpolyadenylation sites; other orders and/or orientations of themonocistronic and bicistronic genes; other anti-repressor elements orparts, derivations, and/or analogues thereof; other vector systems fordelivery of the inventive DNA molecules into eukaryotic host cells; andapplications of the inventive method to other transgenic systems.

EXAMPLES Example 1 STAR Elements and Two-Step Selection Improve thePredictability of Transgene Expression

Improved transgene expression for heterologous protein production isprovided by using a two-step antibiotic selection procedure. Thetwo-step procedure increases the predictability of finding recombinanthost cell lines that express the transgene to high levels, thusincreasing the yield of the heterologous protein.

Materials and Methods

Plasmid Construction

The pSDH-SIB/Z and pSDH-GIB/Z families of plasmids were constructed asfollows: The zeocin selectable marker was recovered by polymerase chainreaction (PCR) amplification from plasmid pEM7/zeo (Invitrogen V500-20)using primers E99 and E100 (SEQ ID NOS:186 and 187, respectively) (allPCR primers and mutagenic oligonucleotide sequences are listed in Table1), and cloned directionally into the XbaI and NotI sites of multiplecloning site (MCS) B of pIRES (Clontech 6028-1) to create pIRES-zeo. Theblasticidin selectable marker was recovered by PCR from plasmid pCMV/bsd(Invitrogen V510-20) using primers E84 and E85 (SEQ ID NOS:176 and 177,respectively), and cloned directionally into the XbaI and NotI sitesMCS-B of pIRES to create pIRES-bsd. The SEAP (secreted alkalinephosphatase) reporter gene was recovered by PCR from plasmidpSEAP2-basic (Clontech 6049-1) using primers F11 and E87 (SEQ ID NOS:188and 178, respectively), and cloned directionally into MCS-A of pIRES-zeoand pIRES-bsd to create plasmids pIRES-SEAP-zeo and pIRES-SEAP-bsd. TheGFP reporter gene was recovered from plasmid phr-GFP-1 (Stratagene240059) by restriction digestion with NheI and EcoRI, and ligateddirectionally into MCS-A of pIRES-zeo and pIRES-bsd to create plasmidspIRES-GFP-zeo and pIRES-GFP-bsd. A linker was inserted at thenon-methylated ClaI site of each of these plasmids (downstream of theneomycin resistance marker) to introduce an AgeI site usingoligonucleotides F34 and F35 (SEQ ID NOS:204 and 205, respectively).

The pSDH-Tet vector was constructed by PCR of the luciferase openreading frame from plasmid pREP4-HSF-Luc (van der Vlag et al., 2000)using primers C67 and C68 (SEQ ID NOS:142 and 143, respectively), andinsertion of the SacII/BamHI fragment into SacII/BamHI-digested pUHD10-3(Gossen & Bujard, 1992). The luciferase expression unit was re-amplifiedwith primers C65 and C66 (SEQ ID NOS:140 and 141, respectively), andre-inserted into pUHD10-3 in order to flank it with multiple cloningsites (MCSI and MCSII). An AscI site was then introduced into MCSI bydigestion with EcoRI and insertion of a linker (comprised of annealedoligonucleotides D93 and D94, SEQ ID NOS:158 and 159, respectively). TheCMV promoter was amplified from plasmid pCMV-Bsd with primers D90 andD91 (SEQ ID NOS:156 and 157, respectively), and used to replace theTet-Off promoter in pSDH-Tet by SalI/SacII digestion and ligation tocreate vector pSDH-CMV. The luciferase open reading frame in this vectorwas replaced by SEAP as follows: vector pSDH-CMV was digested with SacIIand BamHI and made blunt; the SEAP open reading frame was isolated frompSEAP-basic by EcoRI/SalI digestion, made blunt and ligated intopSDH-CMV to create vector pSDH-CS. The puromycin resistance gene undercontrol of the SV40 promoter was isolated from plasmid pBabe-Puro(Morgenstern & Land, 1990) by PCR, using primers C81 and C82 (SEQ IDNOS:144 and 145, respectively). This was ligated into vectorpGL3-control (BamHI site removed) (Promega E1741) digested withNcoI/XbaI, to create pGL3-puro. pGL3-puro was digested with BglII/SalIto isolate the SV40-puro resistance gene, which was made blunt andligated into NheI digested, blunt-ended pSDH-CS. The resulting vector,pSDH-CSP, is shown in FIG. 2. STAR18 (SEQ ID NO:18) was inserted intoMCSI and MCSII in two steps, by digestion of the STAR element and thepSDH-CSP vector with an appropriate restriction enzyme, followed byligation. The orientation of the STAR element was determined byrestriction mapping. The identity and orientation of the inserts wereverified by DNA sequence analysis. Sequencing was performed by thedideoxy method (Sanger et al., 1977) using a Beckman CEQ2000 automatedDNA sequencer, according to the manufacturer's instructions. Briefly,DNA was purified from E. coli using QIAprep® Spin Miniprep and PlasmidMidi Kits (QIAGEN® 27106 and 12145, respectively). Cycle sequencing wascarried out using custom oligonucleotides C85, E25, and E42 (SEQ IDNOS:146, 173 and 174, respectively) (Table 1), in the presence of dyeterminators CEQ™ Dye Terminator Cycle Sequencing Kit, Beckman 608000).

pSDH-CSP plasmids containing STAR elements were modified as follows: forreceiving SEAP-IRES-zeo/bsd cassettes, an AgeI site was introduced atthe BglII site by insertion of a linker, using oligonucleotides F32 andF33 (SEQ ID NOS:202 and 203, respectively); for receivingGFP-IRES-zeo/bsd cassettes, an AgeI site was introduced at the Bsu36Isite by insertion of a linker, using oligonucleotides F44 and F45 (SEQID NOS:206 and 207, respectively). The SEAP-IRES-zeo/bsd cassettes wereinserted into the pSDH-CSP-STAR18 plasmid by replacement of theBsu36I/AgeI fragment with the corresponding fragments from thepIRES-SEAP-zeo/bsd plasmids. The GFP-IRES-zeo/bsd cassettes wereinserted into pSDH-CSP-STAR plasmids by replacement of the BglII/AgeIfragment with the corresponding fragments from the pIRES-GFP-zeo/bsdplasmids. The resulting plasmid families, pSDH-SIB/Z and pSDH-GIB/Z, areshown in FIGS. 3A and 3B, respectively.

All cloning steps were carried out following the instructions providedby the manufacturers of the reagents used, according to methods known inthe art (Sambrook et al., 1989).

Transfection and Culture of CHO Cells

The Chinese Hamster Ovary cell line CHO-K1 (ATCC CCL-61) was cultured inHAMS-F12 medium+10% Fetal Calf Serum containing 2 mM glutamine, 100 U/mlpenicillin, and 100 micrograms/ml streptomcyin at 37° C., 5% CO₂. Cellswere transfected with the pSDH-SIZ plasmids using SuperFect® (QIAGEN®)as described by the manufacturer. Briefly, cells were seeded to culturevessels and grown overnight to 70-90% confluence. SuperFect® reagent wascombined with plasmid DNA at a ratio of 6 microliters per microgram(e.g., for a 10 cm Petri dish, 20 micrograms DNA and 120 microlitersSuperFect®) and added to the cells. After overnight incubation thetransfection mixture was replaced with fresh medium, and the transfectedcells were incubated further. After overnight cultivation, cells wereseeded into fresh culture vessels and 500 micrograms/ml neomycin wasadded. Neomycin selection was complete within three to four days. Freshmedium was then added containing zeocin (100 μg/ml) and culturedfurther. Individual clones were isolated after 4-5 days and culturedfurther. Expression of the reporter gene was assessed by measuring SEAPactivity approximately three weeks after transfection.

Secreted Alkaline Phosphatase (SEAP) Assay

SEAP activity (Berger et al., 1988, Henthorn et al., 1988, Kain, 1997,Yang et al., 1997) in the culture media of the clones was determined asdescribed by the manufacturer (Clontech Great EscAPe™ kit #K2041).Briefly, an aliquot of medium was heat inactivated at 65° C., thencombined with assay buffer and CSPD chemiluminescent substrate andincubated at room temperature for ten minutes. The rate of substrateconversion was then determined in a luminometer (Turner 20/20TD). Celldensity was determined by counting trypsinized cells in a Coulter ACT10cell counter.

Results

Transfection of the pSDH-SIZ-STAR18 expression vector consistentlyresults in ˜10-fold more colonies than transfection of the emptypSDH-SIZ vector, presumably due to the increased proportion of primarytransfectants that are able to bring the neomycin resistance gene toexpression. The outcome of a typical experiment is shown in Table 2, inwhich transfection of the empty vector yielded ˜100 G418-resistantcolonies, and transfection of the STAR18 vector yielded ˜1000 colonies.

The expression of the SEAP reporter transgene was compared between theempty pSDH-SIZ vector (hence, without a STAR sequence) and the STAR18vector FIG. 4). The populations of G418-resistant isolates were dividedinto two sets. The first set was cultured with G418 only (one-stepselection). For this set, the inclusion of STAR18 (SEQ ID NO:18) toprotect the transgene from silencing resulted in higher yield ofreporter protein: the maximal level of expression among the 20 clonesanalyzed was 2-3-fold higher than the maximal expression level of cloneswithout the STAR element. The inclusion of STAR18 (SEQ ID NO:18) alsoled increased predictability: more than 25% of the STAR18 clones hadexpression levels greater than or equal to the maximum expression levelobserved in the STARless clones. In this population of STAR18 clones,70% had expression above the background level, while only 50% of theSTARless clones had expression above the background level.

The performance of STAR18 (SEQ ID NO:18) was even better when used in atwo-step selection. The second set of G418-resistant isolates wastreated with zeocin. Clones that survived the two-step selection regimewere assayed for expression of the SEAP reporter transgene. In this casetoo, the STAR18 (SEQ ID NO:18) element increased the yield compared tothe STARless clones by approximately three-fold. The predictability wasalso increased by inclusion of STAR18 (SEQ ID NO:18): ˜80% of thepopulation had expression levels greater than the highest-expressingSTARless clone.

When the one-step selection is compared with the two-step selection, itcan be seen that the latter is superior in terms of both yield andpredictability. In fact with two-step selection, no clones appear withbackground levels of expression. This is due to the requirement imposedon clones that survive zeocin selection that they have high levels oftranscription of the bicistronic SEAP-zeocin gene. As indicated in Table2, the elimination of low-producing clones by the second antibioticselection step increases the predictability of finding high-producingclones; when STAR18 (SEQ ID NO:18) is included in the expression unit,this increased predictability is improved from three-fold tothirty-fold. In summary, when STAR elements are used in combination withtwo-step antibiotic selection, the predictability of finding clones withhigh yields of a transgene is dramatically improved. Application of thisincreased predictability to two or more transgenes simultaneously willsignificantly increase the likelihood of finding clones that have highyields of multimeric proteins.

Example 2 Simultaneous Expression of Two Proteins is Improved byTwo-Step Selection and STAR Elements

Improved expression of heterologous multimeric proteins such asantibodies is also provided. This example demonstrates that thecombination of STAR elements and two-step antibiotic selection improvesthe predictability of establishing recombinant host cell lines thatexpress balanced and proportional amounts of two heterologouspolypeptides at high yields. This method of the invention is applicablein practice to multimeric proteins such as antibodies. It isdemonstrated in this example using two reporter proteins, secretedalkaline phosphatase (SEAP) and green fluorescent protein (GFP).

Materials and Methods

Plasmids

The pSDH-SIB/Z and pSDH-GIB/Z families of plasmids described in Example1 are used. Cloning of STAR elements x and y, transfection and cultureof host cells, and SEAP assay are described in Example 1. The assay forGFP is performed according to the manufacturer's instructions.

Results

Results show an increased number of clones wherein the two reporterproteins are both expressed. Moreover, expression was balanced in manyof such clones.

Example 3 General-Purpose Vectors for Simultaneous Expression ofMultiple Polypeptides

The expression system tested and validated in Example 1 has beenmodified to facilitate its application to any polypeptide that ispreferably co-expressed with another polypeptide or polypeptides in ahost cell, for example, the heavy and light chains of recombinantantibodies. It is designed for easy and rapid construction of theexpression units. This improved system is described in this example.

Materials and Methods

Plasmids

The construction of the plasmids PP1 to PP5 is described below, andtheir map is shown in FIG. 5. Plasmid pd2EGFP (Clontech 6010-1) wasmodified by insertion of a linker at the BsiWI site to yieldpd2EGFP-link. The linker (made by annealing oligonucleotides F25 andF26, SEQ ID NOS:200 and 201, respectively) introduces sites for thePacI, BglII, and EcoRV restriction endonucleases. This creates themultiple cloning site MCSII for insertion of STAR elements. Then primersF23 and F24 (SEQ ID NOS:198 and 199, respectively) were used to amplifya region of 0.37 kb from pd2EGFP, which was inserted into the BglII siteof pIRES (Clontech 6028-1) to yield pIRES-stuf. This introduces sitesfor the AscI and SwaI restriction endonucleases at MCSI, and acts as a“stuffer fragment” to avoid potential interference between STAR elementsand adjacent promoters. pIRES-stuf was digested with BglII and FspI toliberate a DNA fragment composed of the stuffer fragment, the CMVpromoter, the IRES element (flanked by multiple cloning sites MCS A andMCS B), and the SV40 polyadenylation signal. This fragment was ligatedwith the vector backbone of pd2EGFP-link produced by digestion withBamHI and StuI, to yield pd2IRES-link.

The open reading frames of the zeocin-, neomycin, orpuromycin-resistance genes were inserted into the BamHI/NotI sites ofMCS B in pd2IRES-link as follows: the zeocin-resistance ORF wasamplified by PCR with primers F18 and E100 (SEQ ID NOS:193 and 187,respectively) from plasmid pEM7/zeo, digested with BamHI and NotI, andligated with BamHI/NotI-digested pd2IRES-link to yield pd2IRES-link-zeo.The neomycin-resistance ORF was amplified by PCR with primers F19 andF20 (SEQ ID NOS:194 and 195, respectively) from pIRES, digested withBamHI and NotI, and ligated with BamHI/NotI-digested pd2IRES-link toyield pd2IRES-link-neo. The puromycin-resistance ORF was amplified byPCR with primers F21 and F22 (SEQ ID NOS:196 and 197, respectively) fromplasmid pBabe-Puro (Morgenstern & Land, 1990), digested with BamHI andNotI, and ligated with BamHI/NotI-digested pd2IRES-link to yieldpd2IRES-link-puro.

The GFP reporter ORF was introduced into pd2IRES-link-puro byamplification of phr-GFP-1 with primers F16 and F17 (SEQ ID NOS:191 and192, respectively), and insertion of the EcoRI-digested GFP cassetteinto the EcoRI site in MCS A of the pd2IRES-link-puro plasmid, to yieldplasmid PP1 (FIG. 5A). Correct orientation was verified by restrictionmapping. The SEAP reporter ORF was introduced into pd2IRES-link-zeo andpd2IRES-link-neo by PCR amplification of pSEAP2-basic with primers F14and F15 (SEQ ID NOS:189 and 190, respectively), and insertion of theEcoRI-digested SEAP cassette into the EcoRI sites in MCS A of theplasmids pd2IRES-link-zeo (to yield plasmid PP2, FIG. 5B) andpd2IRES-link-neo (to yield plasmid PP3, FIG. 5C). Correct orientationwas verified by restriction mapping.

Plasmids PP1, PP2 and PP3 contain a bicistronic gene for expression of areporter protein and an antibiotic resistance marker. In order to carryout two-step antibiotic selection with separate antibiotics, amonocistronic resistance marker was introduced as follows: pIRES-stufwas digested with ClaI, made blunt with Klenow enzyme, and digestedfurther with BglII. This liberated a DNA fragment composed of thestuffer fragment, the CMV promoter, the IRES element (flanked bymultiple cloning sites MCS A and MCS B), the SV40 polyadenylationsignal, and the neomycin resistance marker under control of the SV40promoter. This fragment was ligated with the vector backbone ofpd2EGFP-link produced by digestion with BamHI and StuI, to yieldpd2IRES-link-neo. Then as described above the GFP and puro cassetteswere introduced to yield PP4 (FIG. 5D), and the SEAP and zeo cassetteswere introduced to yield PP5 (FIG. 5E).

Example 4 Predictability and Yield are Improved by Application of STARElements in Expression Systems

STAR elements function to block the effect of transcriptional repressioninfluences on transgene expression units. These repression influencescan be due to heterochromatin (“position effects” (Boivin & Dura, 1998))or to adjacent copies of the transgene (“repeat-induced gene silencing”(Garrick et al., 1998)). Two of the benefits of STAR elements forprotein production are increased predictability of findinghigh-expressing primary recombinant host cells, and increased yieldduring production cycles. These benefits are illustrated in thisexample.

Materials and Methods

Construction of the pSDH vectors and STAR-containing derivatives: ThepSDH-Tet vector was constructed by polymerase chain reactionamplification (PCR) of the luciferase open reading frame from plasmidpREP4-HSF-Luc (van der Vlag et al., 2000) using primers C67 and C68 (SEQID NOS:142 and 143, respectively) (all PCR primers and mutagenicoligonucleotides are listed in Table 1), and insertion of theSacII/BamHI fragment into SacII/BamHI-digested pUHD10-3 (Gossen &Bujard, 1992). The luciferase expression unit was re-amplified withprimers C65 and C66 (SEQ ID NOS:140 and 141, respectively), andre-inserted into pUHD10-3 in order to flank it with two multiple cloningsites (MCSI and MCSII). An AscI site was then introduced into MCSI bydigestion with EcoRI and insertion of a linker (comprised of annealedoligonucleotides D93 and D94, SEQ ID NOS:158 and 159, respectively). TheCMV promoter was amplified from plasmid pCMV-Bsd (Invitrogen K510-01)with primers D90 and D91 (SEQ ID NOS:156 and 157, respectively), andused to replace the Tet-Off promoter in pSDH-Tet by SalI/SacII digestionand ligation to create vector pSDH-CMV. The luciferase open readingframe in this vector was replaced by SEAP (Secreted AlkalinePhosphatase) as follows: vector pSDH-CMV was digested with SacII andBamHI and made blunt; the SEAP open reading frame was isolated frompSEAP-basic (Clontech 6037-1) by EcoRI/SalI digestion, made blunt andligated into pSDH-CMV to create vector pSDH-CS. The puromycin resistancegene under control of the SV40 promoter was isolated from plasmidpBabe-Puro (Morgenstern & Land, 1990) by PCR, using primers C81 and C82(SEQ ID NOS:144 and 145, respectively). This was ligated into vectorpGL3-control (BamHI site removed) (Promega E1741) digested withNcoI/XbaI, to create pGL3-puro. pGL3-puro was digested with BglII/SalIto isolate the SV40-puro resistance gene, which was made blunt andligated into NheI digested, blunt-ended pSDH-CS. The resulting vector,pSDH-CSP, is shown in FIG. 6. All cloning steps were carried outfollowing the instructions provided by the manufacturers of thereagents, according to methods known in the art (Sambrook et al., 1989).

STAR elements were inserted into MCSI and MCSII in two steps, bydigestion of the STAR element and the pSDH-CSP vector with anappropriate restriction enzyme, followed by ligation. The orientation ofSTAR elements in recombinant pSDH vectors was determined by restrictionmapping. The identity and orientation of the inserts were verified byDNA sequence analysis. Sequencing was performed by the dideoxy method(Sanger et al., 1977) using a Beckman CEQ™ 2000 automated DNA sequencer,according to the manufacturer's instructions. Briefly, DNA was purifiedfrom E. coli using QIAprep® Spin Miniprep and Plasmid Midi Kits (QIAGEN®27106 and 12145, respectively). Cycle sequencing was carried out usingcustom oligonucleotides C85, E25, and E42 (SEQ ID NOS:146, 173 and 174,respectively) (Table 1), in the presence of dye terminators (CEQ™ DyeTerminator Cycle Sequencing Kit, Beckman 608000).

Transfection and Culture of CHO Cells with pSDH Plasmids

The Chinese Hamster Ovary cell line CHO-K1 (ATCC CCL-61) was cultured inHAMS-F12 medium+10% Fetal Calf Serum containing 2 mM glutamine, 100 U/mlpenicillin, and 100 micrograms/ml streptomcyin at 3° C., 5% CO₂. Cellswere transfected with the pSDH-CSP vector, and its derivativescontaining STAR6 (SEQ ID NO:6) or STAR49 (SEQ ID NO:49) in MCSI andMCSII, using SuperFect® (QIAGEN®) as described by the manufacturer.Briefly, cells were seeded to culture vessels and grown overnight to70-90% confluence. SuperFect® reagent was combined with plasmid DNA(linearized in this example by digestion with PvuI) at a ratio of 6microliters per microgram (e.g., for a 10 cm Petri dish, 20 microgramsDNA and 120 microliters SuperFect®) and added to the cells. Afterovernight incubation the transfection mixture was replaced with freshmedium, and the transfected cells were incubated further. Afterovernight cultivation, 5 micrograms/ml puromycin was added. Puromycinselection was complete in two weeks, after which time individualpuromycin resistant CHO/pSDH-CSP clones were isolated at random andcultured further.

Secreted Alkaline Phosphatase (SEAP) Assay

SEAP activity (Berger et al., 1988, Henthorn et al., 1988, Kain, 1997,Yang et al., 1997) in the culture medium of CHO/pSDH-CSP clones wasdetermined as described by the manufacturer (Clontech Great EscAPe™ kit#K2041). Briefly, an aliquot of medium was heat inactivated at 65° C.,then combined with assay buffer and CSPD chemiluminescent substrate andincubated at room temperature for ten minutes. The rate of substrateconversion was then determined in a luminometer (Turner 20/20TD). Celldensity was determined by counting trypsinized cells in a Coulter ACT10cell counter.

Transfection and Culture of U-2 OS Cells with pSDH Plasmids

The human osteosarcoma U-2 OS cell line (ATCC #HTB-96) was cultured inDulbecco's Modified Eagle Medium+10% Fetal Calf Serum containingglutamine, penicillin, and streptomycin (supra) at 37° C., 5% CO₂. Cellswere co-transfected with the pSDH-CMV vector, and its derivativescontaining STAR6 (SEQ ID NO:6) or STAR8 (SEQ ID NO:8) in MCSI and MCSII,(along with plasmid pBabe-Puro) using SuperFect® (supra). Puromycinselection was complete in two weeks, after which time individualpuromycin resistant U-2 OS/pSDH-CMV clones were isolated at random andcultured further.

Luciferase Assay

Luciferase activity (Himes & Shannon, 2000) was assayed in resuspendedcells according to the instructions of the assay kit manufacturer (Roche1669893), using a luminometer (Turner 20/20TD). Total cellular proteinconcentration was determined by the bicinchoninic acid method accordingto the manufacturer's instructions (Sigma B-9643), and used to normalizethe luciferase data.

Results

Recombinant CHO cell clones containing the pSDH-CSP vector, or pSDH-CSPplasmids containing STAR6 (SEQ ID NO:6) or STAR49 (SEQ ID NO:49) (Table6), were cultured for three weeks. The SEAP activity in the culturesupernatants was then determined, and is expressed on the basis of cellnumber (FIG. 7). As can be seen, clones with STAR elements in theexpression units were isolated that express 2-3 fold higher SEAPactivity than clones whose expression units do not include STARelements. Furthermore, the number of STAR-containing clones that expressSEAP activity at or above the maximal activity of the STAR-less clonesis quite high: 25% to 40% of the STAR clone populations exceed thehighest SEAP expression of the pSDH-CSP clones.

Recombinant U-2 OS cell clones containing the pSDH-CMV vector, orpSDH-CMV plasmids containing STAR6 (SEQ ID NO:6) or STAR8 (SEQ ID NO:8)(Table 6), were cultured for three weeks. The luciferase activity in thehost cells was then determined, and is expressed as relative luciferaseunits (FIG. 8), normalized to total cell protein. The recombinant U-2 OSclones with STAR elements flanking the expression units had higheryields than the STAR-less clones: the highest expression observed fromSTAR8 clones was 2-3 fold higher than the expression from STAR-lessclones. STAR6 clones had maximal expression levels five-fold higher thanthe STAR-less clones. The STAR elements conferred greater predictabilityas well: for both STAR elements, 15 to 20% of the clones displayedluciferase expression at levels comparable to or greater than theSTAR-less clone with the highest expression level.

These results demonstrate that, when used with the strong CMV promoter,STAR elements increase the yield of heterologous proteins (luciferaseand SEAP). All three of the STAR elements introduced in this exampleprovide elevated yields. The increased predictability conferred by theSTAR elements is manifested by the large proportion of the clones withyields equal to or greater than the highest yields displayed by theSTAR-less clones.

Example 5 STAR Elements Improve the Stability of Transgene Expression

During cultivation of recombinant host cells, it is common practice tomaintain antibiotic selection. This is intended to preventtranscriptional silencing of the transgene, or loss of the transgenefrom the genome by processes such as recombination. However it isundesirable for production of proteins, for a number of reasons. First,the antibiotics that are used are quite expensive, and contributesignificantly to the unit cost of the product. Second, forbiopharmaceutical use, the protein must be demonstrably pure, with notraces of the antibiotic in the product. One advantage of STAR elementsfor heterologous protein production is that they confer stableexpression on transgenes during prolonged cultivation, even in theabsence of antibiotic selection; this property is demonstrated in thisexample.

Materials and Methods

The U-2 OS cell line was transfected with the plasmid pSDH-Tet-STAR6 andcultivated as described in Example 4. Individual puromycin-resistantclones were isolated and cultivated further in the absence ofdoxycycline. At weekly intervals the cells were transferred to freshculture vessels at a dilution of 1:20. Luciferase activity was measuredat periodic intervals as described in Example 4. After 15 weeks thecultures were divided into two replicates; one replicate continued toreceive puromycin, while the other replicate received no antibiotic forthe remainder of the experiment (25 weeks total).

Results

Table 7 presents the data on luciferase expression by an expression unitflanked with STAR6 (SEQ ID NO:6) during prolonged growth with or withoutantibiotic. As can be seen, the expression of the reporter transgene,luciferase, remains stable in the U-2 OS host cells for the duration ofthe experiment. After the cultures were divided into two treatments(plus antibiotic and without antibiotic) the expression of luciferasewas essentially stable in the absence of antibiotic selection. Thisdemonstrates the ability of STAR elements to protect transgenes fromsilencing or loss during prolonged cultivation. It also demonstratesthat this property is independent of antibiotic selection. Therefore,production of proteins is possible without incurring the costs of theantibiotic or of difficult downstream processing.

Example 6 Minimal Essential Sequences of STAR Elements

STAR elements are isolated from the genetic screen as described herein.The screen uses libraries constructed with human genomic DNA that wassize-fractionated to approximately 0.5-2 kilobases (supra). The STARelements range from 500 to 2361 base pairs (Table 6). It is likely that,for many of the STAR elements that have been isolated, STAR activity isconferred by a smaller DNA fragment than the initially isolated clone.It is useful to determine these minimum fragment sizes that areessential for STAR activity, for two reasons. First, smaller functionalSTAR elements would be advantageous in the design of compact expressionvectors, since smaller vectors transfect host cells with higherefficiency. Second, determining minimum essential STAR sequences permitsthe modification of those sequences for enhanced functionality. Two STARelements have been fine-mapped to determine their minimal essentialsequences.

Materials and Methods:

STAR10 (SEQ ID NO:10) (1167 base pairs) and STAR27 (SEQ ID NO:27) (1520base pairs) have been fine-mapped. They have been amplified by PCR toyield sub-fragments of approximately equal length (FIG. 9 legend). Forinitial testing, these have been cloned into the pSelect vector at theBamHI site, and transfected into U-2 OS/Tet-Off/LexA-HP1 cells. Theconstruction of the host strains has been described (van der Vlag etal., 2000). Briefly, they are based on the U-2 OS human osteosarcomacell line (American Type Culture Collection HTB-96). U-2 OS is stablytransfected with the pTet-Off plasmid (Clontech K1620-A), encoding aprotein chimera consisting of the Tet-repressor DNA binding domain andthe VP16 transactivation domain. The cell line is subsequently stablytransfected with fusion protein genes containing the LexA DNA bindingdomain, and the coding regions of either HP1 or HPC2 (two DrosophilaPolycomb group proteins that repress gene expression when tethered toDNA). The LexA-repressor genes are under control of the Tet-Offtranscriptional regulatory system (Gossen and Bujard, 1992). Afterselection for hygromycin resistance, LexA-HP1 was induced by loweringthe doxycycline concentration. Transfected cells were then incubatedwith zeocin to test the ability of the STAR fragments to protect theSV40-Zeo expression unit from repression due to LexA-HP1 binding.

Results

In this experiment STAR10 (SEQ ID NO:10) and STAR27 (SEQ ID NO:27)confer good protection against gene silencing, as expected (FIG. 9).This is manifested by robust growth in the presence of zeocin.

Of the three STAR10 (SEQ ID NO:10) sub-fragments, 10A (corresponding insequence to roughly the first 400 nucleotides of SEQ ID NO:10) (˜400base pairs) confers on transfected cells vigorous growth in the presenceof zeocin, exceeding that of the full-length STAR element. Cellstransfected with pSelect constructs containing the other twosub-fragments do not grow in the presence of zeocin. These resultsidentify the ˜400 base pair 10A fragment as encompassing the DNAsequence responsible for the anti-repression activity of STAR10 (SEQ IDNO:10).

STAR27 (SEQ ID NO:27) confers moderate growth in zeocin to transfectedcells in this experiment (FIG. 9). One of the sub-fragments of thisSTAR, 27B (corresponding in sequence to roughly the second 500nucleotides of SEQ ID NO:27) (˜500 base pairs), permits weak growth ofthe host cells in zeocin-containing medium. This suggests that theanti-repression activity of this STAR is partially localized onsub-fragment 27B (corresponding in sequence to roughly the first 500nucleotides of SEQ ID NO:27), but full activity requires sequences from27A (corresponding in sequence to roughly the first 500 nucleotides ofSEQ ID NO:27) and/or 27C (corresponding in sequence to roughly the third500 nucleotides of SEQ ID NO:27) (each ˜500 base pairs) as well.

Example 7 STAR Elements Function in Diverse Strains of CulturedMammalian Cells

The choice of host cell line for (heterologous) protein expression is acritical parameter for the quality, yield, and unit cost of the protein.Considerations such as post-translational modifications, secretorypathway capacity, and cell line immortality dictate the appropriate cellline for a particular biopharmaceutical production system. For thisreason, the advantages provided by STAR elements in terms of yield,predictability, and stability should be obtainable in diverse celllines. This was tested by comparing the function of STAR6 (SEQ ID NO:6)in the human U-2 OS cell line in which it was originally cloned, and theCHO cell line which is widely applied in biotechnology.

Materials and Methods:

The experiments of Example 4 are referred to.

Results

The expression of the SEAP reporter gene in CHO cells is presented inFIG. 7; the expression of the luciferase reporter gene in U-2 OS cellsis presented in FIG. 8. By comparison of the results of these twoexperiments, it is apparent that the STAR6 (SEQ ID NO:6) element isfunctional in both cell lines: reporter gene expression was morepredictable in both of them, and clones of each cell line displayedhigher yields, when the reporter gene was shielded from position effectsby STAR6 (SEQ ID NO:6). These two cell lines are derived from differentspecies (human and hamster) and different tissue types (bone and ovary),reflecting the broad range of host cells in which this STAR element canbe utilized in improving heterologous protein expression.

Example 8 STAR Elements Function in the Context of VariousTranscriptional Promoters

Transgene transcription is achieved by placing the transgene openreading frame under control of an exogenous promoter. The choice ofpromoter is influenced by the nature of the (heterologous) protein andthe production system. In most cases, strong constitutive promoters arepreferred because of the high yields they can provide. Some viralpromoters have these properties; the promoter/enhancer of thecytomegalovirus immediate early gene (“CMV promoter”) is generallyregarded as the strongest promoter in common biotechnological use(Boshart et al., 1985, Doll et al., 1996, Foecking & Hofstetter, 1986).The simian virus SV40 promoter is also moderately strong (Boshart etal., 1985, Foecking & Hofstetter, 1986) and is frequently used forectopic expression in mammalian cell vectors. The Tet-Off promoter isinducible: the promoter is repressed in the presence of tetracycline orrelated antibiotics (doxycycline is commonly used) in cell-lines whichexpress the tTA plasmid (Clontech K1620-A), and removal of theantibiotic results in transcriptional induction (Deuschle et al., 1995,Gossen & Bujard, 1992, Izumi & Gilbert, 1999, Umana et al., 1999).

Materials and Methods:

The construction of the pSDH-Tet and pSDH-CMV vectors is described inExample 4. pSDH-SV40 is, amongst others, derived from pSelect-SV40-zeo.The selection vector for STAR elements, pSelect-SV40-zeo is constructedas follows: the pREP4 vector (Invitrogen V004-50) is used as the plasmidbackbone. It provides the Epstein Barr oriP origin of replication andEBNA-1 nuclear antigen for high-copy episomal replication in primatecell lines; the hygromycin resistance gene with the thymidine kinasepromoter and polyadenylation site, for selection in mammalian cells; andthe ampicillin resistance gene and colE1 origin of replication formaintenance in Escherichia coli. The vector contains four consecutiveLexA operator sites between XbaI and NheI restriction sites (Bunker andKingston, 1994). Embedded between the LexA operators and the NheI siteis a polylinker consisting of the following restriction sites:HindIII-AscI-BamHI-AscI-HindIII. Between the NheI site and a SalI siteis the zeocin resistance gene with the SV40 promoter and polyadenylationsite, derived from pSV40/Zeo (Invitrogen V502-20); this is theselectable marker for the STAR screen.

pSDH-SV40 was constructed by PCR amplification of the SV40 promoter(primers D41 and D42) from plasmid pSelect-SV40-Zeo, followed bydigestion of the PCR product with SacII and SalI. The pSDH-CMV vectorwas digested with SacII and SalI to remove the CMV promoter, and thevector and SV40 fragment were ligated together to create pSDH-SV40.STAR6 (SEQ ID NO:6) was cloned into MCSI and MCSII as described inExample 4. The plasmids pSDH-Tet, pSDH-Tet-STAR6, pSDH-Tet-STAR7,pSDH-SV40 and pSDH-SV40-STAR6 were co-transfected with pBabe-Puro intoU-2 OS using SuperFect as described by the manufacturer. Cellcultivation, puromycin selection, and luciferase assays were carried outas described in Example 4.

Results

FIGS. 9, 11, and 12 compare the expression of the luciferase reportergene from three different promoters: two strong and constitutive viralpromoters (CMV and SV40), and the inducible Tet-Off promoter. All threepromoters were tested in the context of the STAR6 (SEQ ID NO:6) elementin U-2 OS cells. The results demonstrate that the yield andpredictability from all three promoters are increased by STAR6 (SEQ IDNO:6). As described in Examples 4 and 7, STAR6 (SEQ ID NO:6) isbeneficial in the context of the CMV promoter (FIG. 8). Similarimprovements are seen in the context of the SV40 promoter (FIG. 10): theyield from the highest-expressing STAR6 clone is 2-3 fold greater thanthe best pSDH-SV40 clones, and six STAR clones (20% of the population)have yields higher than the best STAR-less clones. In the context of theTet-Off promoter under inducing (low doxycycline) concentrations, STAR6(SEQ ID NO:6) also improves the yield and predictability of transgeneexpression (FIG. 11): the highest-expressing STAR6 clone has a 20-foldhigher yield than the best pSDH-Tet clone, and nine STAR6 clones (35% ofthe population) have yields higher than the best STAR-less clone. It isconcluded that this STAR element is versatile in itstransgene-protecting properties, since it functions in the context ofvarious biotechnologically useful promoters of transcription.

Example 9 STAR Element Function can be Directional

While short nucleic acid sequences can be symmetrical (e.g.,palindromic), longer naturally-occurring sequences are typicallyasymmetrical. As a result, the information content of nucleic acidsequences is directional, and the sequences themselves can be describedwith respect to their 5′ and 3′ ends. The directionality of nucleic acidsequence information affects the arrangement in which recombinant DNAmolecules are assembled using standard cloning techniques known in theart (Sambrook et al., 1989). STAR elements are long, asymmetrical DNAsequences, and have a directionality based on the orientation in whichthey were originally cloned in the pSelect vector. In the examples givenabove, using two STAR elements in pSDH vectors, this directionality waspreserved. This orientation is described as the native or 5′-3′orientation, relative to the zeocin resistance gene (see FIG. 12). Inthis example, the importance of directionality for STAR function istested in the pSDH-Tet vector. Since the reporter genes in the pSDHvectors are flanked on both sides by copies of the STAR element ofinterest, the orientation of each STAR copy must be considered. Thisexample compares the native orientation with the opposite orientation(FIG. 12).

Materials and Methods:

The STAR66 (SEQ ID NO:66) element was cloned into pSDH-Tet as describedin Example 4. U-2 OS cells were co-transfected with plasmidspSDH-Tet-STAR66-native and pSDH-Tet-STAR66-opposite, and cultivated asdescribed in Example 4. Individual clones were isolated and cultivated;the level of luciferase expression was determined as described (supra).

Results

The results of the comparison of STAR66 (SEQ ID NO:66) activity in thenative orientation and the opposite orientation are shown in FIG. 13.When STAR66 (SEQ ID NO:66) is in the opposite orientation, the yield ofonly one clone is reasonably high (60 luciferase units). In contrast,the yield of the highest-expressing clone when STAR66 (SEQ ID NO:66) isin the native orientation is considerably higher (100 luciferase units),and the predictability is much higher as well: seven clones of thenative-orientation population (30%) express luciferase above the levelof the highest-expressing clone from the opposite-orientationpopulation, and 15 of the clones in the native-orientation population(60%) express luciferase above ten relative luciferase units.

Therefore, it is demonstrated that STAR66 (SEQ ID NO:66) function isdirectional.

Example 10 Transgene Expression in the Context of STAR Elements is CopyNumber-Dependent

Transgene expression units for (heterologous) protein expression aregenerally integrated into the genome of the host cell to ensure stableretention during cell division. Integration can result in one ormultiple copies of the expression unit being inserted into the genome;multiple copies may or may not be present as tandem arrays. Theincreased yield demonstrated for transgenes protected by STAR elements(supra) suggests that STAR elements are able to permit the transgeneexpression units to function independently of influences ontranscription associated with the site of integration in the genome(independence from position effects (Boivin & Dura, 1998). It suggestsfurther that the STAR elements permit each expression unit to functionindependently of neighboring copies of the expression unit when they areintegrated as a tandem array (independence from repeat-induced genesilencing (Garrick et al., 1998)). Copy number-dependence is determinedfrom the relationship between transgene expression levels and copynumber, as described in the example below.

Materials and Methods:

U-2 OS cells were co-transfected with pSDH-Tet-STAR10 and cultivatedunder puromycin selection as described (supra). Eight individual cloneswere isolated and cultivated further. Then cells were harvested, and oneportion was assayed for luciferase activity as described (supra). Theremaining cells were lysed and the genomic DNA purified using theDNeasy® Tissue Kit (QIAGEN® 69504) as described by the manufacturer. DNAsamples were quantitated by UV spectrophotometry. Three micrograms ofeach genomic DNA sample were digested with PvuII and XhoI overnight asdescribed by the manufacturer (New England Biolabs), and resolved byagarose gel electrophoresis. DNA fragments were transferred to a nylonmembrane as described (Sambrook et al., 1989), and hybridized with aradioactively labeled probe to the luciferase gene (isolated fromBamHI/SacII-digested pSDH-Tet). The blot was washed as described(Sambrook et al., 1989) and exposed to a phosphorimager screen (PersonalF/X, BioRad). The resulting autoradiogram (FIG. 14) was analyzed bydensitometry to determine the relative strength of the luciferase DNAbands, which represents the transgene copy number.

Results

The enzyme activities and copy numbers (DNA band intensities) ofluciferase in the clones from the pSDH-Tet-STAR10 clone population isshown in FIG. 15. The transgene copy number is highly correlated withthe level of luciferase expression in these pSDH-Tet-STAR10 clones(r=0.86). This suggests that STAR10 (SEQ ID NO:10) confers copynumber-dependence on the transgene expression units, making transgeneexpression independent of other transgene copies in tandem arrays, andindependent of gene-silencing influences at the site of integration.

Example 11 STAR Elements Function as Enhancer Blockers But Not Enhancers

Gene promoters are subject to both positive and negative influences ontheir ability to initiate transcription. An important class of elementsthat exert positive influences are enhancers. Enhancers arecharacteristically able to affect promoters even when they are locatedfar away (many kilobase pairs) from the promoter. Negative influencesthat act by heterochromatin formation (e.g., Polycomb group proteins)have been described above, and these are the target of STAR activity.The biochemical basis for enhancer function and for heterochromatinformation is fundamentally similar, since they both involve binding ofproteins to DNA. Therefore, it is important to determine whether STARelements are able to block positive influences as well as negativeinfluences, in other words, to shield transgenes from genomic enhancersin the vicinity of the site of integration. The ability to shieldtransgenes from enhancer activity ensures stable and predictableperformance of transgenes in biotechnological applications. This exampleexamines the performance of STAR elements in an enhancer-blocking assay.

Another feature of STAR activity that is important to their function isthe increased yield they confer on transgenes (Example 4). STARs areisolated on the basis of their ability to maintain high levels of zeocinexpression when heterochromatin-forming proteins are bound adjacent tothe candidate STAR elements. High expression is predicted to occurbecause STARs are anticipated to block the spread of heterochromatininto the zeocin expression unit. However, a second scenario is that theDNA fragments in, zeocin-resistant clones contain enhancers. Enhancershave been demonstrated to have the ability to overcome the repressiveeffects of Polycomb-group proteins such as those used in the method ofthe STAR screen (Zink & Paro, 1995). Enhancers isolated by thisphenomenon would be considered false positives, since enhancers do nothave the properties claimed here for STARs. In order to demonstrate thatSTAR elements are not enhancers, they have been tested in an enhancerassay.

The enhancer-blocking assay and the enhancer assay are methodologicallyand conceptually similar. The assays are shown schematically in FIG. 16.The ability of STAR elements to block enhancers is performed using theE47/E-box enhancer system. The E47 protein is able to activatetranscription by promoters when it is bound to an E-box DNA sequencelocated in the vicinity of those promoters (Quong et al., 2002). E47 isnormally involved in regulation of B and T lymphocyte differentiation(Quong et al., 2002), but it is able to function in diverse cell typeswhen expressed ectopically (Petersson et al., 2002). The E-box is apalindromic DNA sequence, CANNTG (SEQ ID NO:120) (Knofler et al., 2002).In the enhancer-blocking assay, an E-box is placed upstream of aluciferase reporter gene (including a minimal promoter) in an expressionvector. A cloning site for STAR elements is placed between the E-box andthe promoter. The E47 protein is encoded on a second plasmid. The assayis performed by transfecting both the E47 plasmid and the luciferaseexpression vector into cells; the E47 protein is expressed and binds tothe E-box, and the E47/E-box complex is able to act as an enhancer. Whenthe luciferase expression vector does not contain a STAR element, theE47/E-box complex enhances luciferase expression (FIG. 16A, situation1). When STAR elements are inserted between the E-box and the promoter,their ability to block the enhancer is demonstrated by reducedexpression of luciferase activity (FIG. 16A, situation 2); if STARscannot block enhancers, luciferase expression is activated (FIG. 16A,situation 3).

The ability of STAR elements to act as enhancers utilizes the sameluciferase expression vector. In the absence of E47, the E-box itselfdoes not affect transcription. Instead, enhancer behavior by STARelements will result in activation of luciferase transcription. Theassay is performed by transfecting the luciferase expression vectorwithout the E47 plasmid. When the expression vector does not containSTAR elements, luciferase expression is low (FIG. 16B, situation 1). IfSTAR elements do not have enhancer properties, luciferase expression islow when a STAR element is present in the vector (FIG. 16B, situation2). If STAR elements do have enhancer properties, luciferase expressionwill be activated in the STAR-containing vectors (FIG. 16B, situation3).

Materials and Methods:

The luciferase expression vector was constructed by inserting the E-boxand a human alkaline phosphatase minimal promoter from plasmidmu-E5+E2×6-cat(x) (Ruezinsky et al., 1991) upstream of the luciferasegene in plasmid pGL3-basic (Promega E1751), to createpGL3-E-box-luciferase (gift of W. Romanow). The E47 expression plasmidcontains the E47 open reading frame under control of a beta-actinpromoter in the pHBAPr-1-neo plasmid; E47 in constitutively expressedfrom this plasmid (gift of W. Romanow).

STAR elements 1, 2, 3, 6, 10, 11, 18, and 27 (SEQ ID NOS:1, 2, 3, 6, 10,11, 18 and 27, respectively) have been cloned into the luciferaseexpression vector. Clones containing the Drosophila scs element and thechicken beta-globin HS4-6x core (“HS4”) element have been included aspositive controls (they are known to block enhancers, and to have nointrinsic enhancer properties (Chung et al., 1993, Kellum & Schedl,1992)), and the empty luciferase expression vector has been included asa negative control. All assays were performed using the U-2 OS cellline. In the enhancer-blocking assay, the E47 plasmid was co-transfectedwith the luciferase expression vectors (empty vector, or containing STARor positive-control elements). In the enhancer assay, the E47 plasmidwas co-transfected with STARless luciferase expression vector as apositive control for enhancer activity; all other samples received amock plasmid during co-transfection. The transiently transfected cellswere assayed for luciferase activity 48 hours after plasmid transfection(supra). The luciferase activity expressed from a plasmid containing noE-box or STAR/control elements was subtracted, and the luciferaseactivities were normalized to protein content as described (supra).

Results

FIG. 17 shows the results of the enhancer-blocking assay. In the absenceof STAR elements (or the known enhancer-blocking elements scs and HS4),the E47/E-box enhancer complex activates expression of luciferase(“vector”); this enhanced level of expression has been normalized to100. Enhancer activity is blocked by all STAR elements tested. Enhanceractivity is also blocked by the HS4 and scs elements, as expected (Bellet al., 2001, Gerasimova & Corces, 2001). These results demonstrate thatin addition to their ability to block the spreading of transcriptionalsilencing (negative influences), STAR elements are able to block theaction of enhancers (positive influences).

FIG. 18 shows the results of the enhancer assay. The level of luciferaseexpression due to enhancement by the E47/E-box complex is set at 100(“E47”). By comparison, none of the STAR elements bring aboutsignificant activation of luciferase expression. As expected, the scsand HS4 elements also do not bring about activation of the reportergene. Therefore, it is concluded that at least the tested STAR elementsdo not possess enhancer properties.

Example 12 STAR Elements are Conserved Between Mouse and Human

BLAT analysis of the STAR DNA sequence against the human genome database(WorldWideWeb.genome.ucsc.edu/cgi-bin/hgGateway) reveals that some ofthese sequences have high sequence conservation with other regions ofthe human genome. These duplicated regions are candidate STAR elements;if they do show STAR activity, they would be considered paralogs of thecloned STARs (two genes or genetic elements are said to be paralogous ifthey are derived from a duplication event (Li, 1997)).

BLAST analysis of the human STARs against the mouse genome(WorldWideWeb.www.ensembl.org/Mus_musculus/blastview) also revealsregions of high sequence conservation between mouse and human. Thissequence conservation has been shown for fragments of 15 out of the 65human STAR elements. The conservation ranges from 64% to 89%, overlengths of 141 base pairs to 909 base pairs (Table 8). These degrees ofsequence conservation are remarkable and suggest that these DNAsequences may confer STAR activity within the mouse genome as well. Someof the sequences from the mouse and human genomes in Table 8 could bestrictly defined as orthologs (two genes or genetic elements are said tobe orthologous if they are derived from a speciation event (Li, 1997)).For example, STAR6 (SEQ ID NO:6) is between the SLC8A1 and HAAO genes inboth the human and mouse genomes. In other cases, a cloned human STARhas a paralog within the human genome, and its ortholog has beenidentified in the mouse genome. For example, STAR3a (SEQ ID NO:3) is afragment of the 15q11.2 region of human chromosome 15. This region is96.9% identical (paralogous) with a DNA fragment at 5q33.3 on humanchromosome 5, which is near the IL12B interleukin gene. These human DNAsshare approximately 80% identity with a fragment of the 11B2 region onmouse chromosome 11. The 11B2 fragment is also near the (mouse) IL12Binterleukin gene. Therefore, STAR3a (SEQ ID NO:3) and the mouse 11B2fragment can be strictly defined as paralogs.

In order to test the hypothesis that STAR activity is shared betweenregions of high sequence conservation in the mouse and human genome, oneof the human STARs with a conserved sequence in mouse, STAR18 (SEQ IDNO:18), has been analyzed in greater detail. The sequence conservationin the mouse genome detected with the original STAR18 clone extendsleftward on human chromosome 2 for about 500 base pairs (FIG. 19; leftand right relate to the standard description of the arms of chromosome2). In this example, we examine whether the region of sequenceconservation defines a “naturally occurring” STAR element in human thatis more extensive in length than the original clone. We also examinewhether the STAR function of this STAR element is conserved betweenmouse and human.

Materials and Methods

The region of mouse/human sequence conservation around STAR 18 (SEQ IDNO:18) was recovered from human BAC clone RP11-387A1 by PCRamplification, in three fragments: the entire region (primers E93 andE94, SEQ ID NOS:180 and 181, respectively), the leftward half (primersE93 and E92, SEQ ID NOS:180 and 179, respectively), and the rightwardhalf (primers E57 and E94, SEQ ID NOS:175 and 181, respectively). Thecorresponding fragments from the homologous mouse region were recoveredfrom BAC clone RP23-400H17 in the same fashion (primers E95 (SEQ IDNO:182) and E98 (SEQ ID NO:185), E95 (SEQ ID NO:182) and E96 (SEQ IDNO:183), and E97 (SEQ ID NO:184) and E98 (SEQ ID NO:185), respectively).All fragments were cloned into the pSelect vector and transfected into aU-2 OS/Tet-Off/LexA-HP1 cell line (supra). Following transfection,hygromycin selection was carried out to select for transfected cells.The LexA-HP1 protein was induced by lowering the doxycyclineconcentration, and the ability of the transfected cells to withstand theantibiotic zeocin (a measure of STAR activity) was assessed bymonitoring cell growth.

Results

The original STAR18 clone was isolated from Sau3AI digested human DNAligated into the pSelect vector on the basis of its ability to preventsilencing of a zeocin resistance gene. Alignment of the human STAR18clone (497 base pairs) with the mouse genome revealed high sequencesimilarity (72%) between the orthologous human and mouse STAR18 (SEQ IDNO:18) regions. It also uncovered high similarity (73%) in the regionextending for 488 base pairs immediately leftwards of the Sau3AI sitethat defines the left end of the cloned region (FIG. 21). Outside theseregions the sequence similarity between human and mouse DNA drops below60%.

As indicated in FIG. 19, both the human STAR18 (SEQ ID NO:18) and themouse STAR18 elements confer survival on zeocin to host cells expressingthe lexA-HP1 repressor protein. The original 497 base pair STAR18 cloneand its mouse ortholog both confer the ability to grow (FIG. 19, a andd). The adjacent 488 base pair regions of high similarity from bothgenomes also confer the ability to grow, and in fact their growthphenotype is more vigorous than that of the original STAR18 clone (FIG.19, b and e). When the entire region of sequence similarity was tested,these DNAs from both mouse and human confer growth, and the growthphenotype is more vigorous than the two sub-fragments (FIG. 19, c andf). These results demonstrate that the STAR activity of human STAR18(SEQ ID NO:18) is conserved in its ortholog from mouse. The highsequence conservation between these orthologous regions is particularlynoteworthy because they are not protein-coding sequences, leading to theconclusion that they have some regulatory function that has preventedtheir evolutionary divergence through mutation.

This analysis demonstrates that cloned STAR elements identified by theoriginal screening program may in some cases represent partial STARelements, and that analysis of the genomic DNA in which they areembedded can identify sequences with stronger STAR activity.

Example 13 STAR Elements Contain Characteristic DNA Sequence Motifs

STAR elements are isolated on the basis of their anti-repressionphenotype with respect to transgene expression. This anti-repressionphenotype reflects underlying biochemical processes that regulatechromatin formation which are associated with the STAR elements. Theseprocesses are typically sequence-specific and result from proteinbinding or DNA structure. This suggests that STAR elements will shareDNA sequence similarity. Identification of sequence similarity amongSTAR elements will provide sequence motifs that are characteristic ofthe elements that have already been identified by functional screens andtests. The sequence motifs will also be useful to recognize and claimnew STAR elements whose functions conform to the claims of this patent.The functions include improved yield and stability of transgenesexpressed in eukaryotic host cells.

Other benefits of identifying sequence motifs that characterize STARelements include: (1) provision of search motifs for prediction andidentification of new STAR elements in genome databases, (2) provisionof a rationale for modification of the elements, and (3) provision ofinformation for functional analysis of STAR activity. Usingbio-informatics, sequence similarities among STAR elements have beenidentified; the results are presented in this example.

Bio-informatic and Statistical Background. Regulatory DNA elementstypically function via interaction with sequence-specific DNA-bindingproteins. Bio-informatic analysis of DNA elements such as STAR elementswhose regulatory properties have been identified, but whose interactingproteins are unknown, requires a statistical approach for identificationof sequence motifs. This can be achieved by a method that detects shortDNA sequence patterns that are over-represented in a set of regulatoryDNA elements (e.g., the STAR elements) compared to a reference sequence(e.g., the complete human genome). The method determines the number ofobserved and expected occurrences of the patterns in each regulatoryelement. The number of expected occurrences is calculated from thenumber of observed occurrences of each pattern in the referencesequence.

The DNA sequence patterns can be oligonucleotides of a given length,e.g., six base pairs. In the simplest analysis, for a 6 base pairoligonucleotide (hexamer) composed of the four nucleotides (A, C, G, andT) there are 4⁶=4096 distinct oligonucleotides (all combinations fromAAAAAA to TTTTTT, SEQ ID NOS:121 and 122, respectively). If theregulatory and reference sequences were completely random and had equalproportions of the A, C, G, and T nucleotides, then the expectedfrequency of each hexamer would be 1/4096 (˜0.00024). However, theactual frequency of each hexamer in the reference sequence is typicallydifferent than this due to biases in the content of G:C base pairs, etc.Therefore, the frequency of each oligonucleotide in the referencesequence is determined empirically by counting, to create a “frequencytable” for the patterns.

The pattern frequency table of the reference sequence is then used tocalculate the expected frequency of occurrence of each pattern in theregulatory element set. The expected frequencies are compared with theobserved frequencies of occurrence of the patterns. Patterns that are“over-represented” in the set are identified; for example, if thehexamer ACGTGA (SEQ ID NO:123) is expected to occur five times in 20kilobase pairs of sequence, but is observed to occur 15 times, then itis three-fold over-represented. Ten of the 15 occurrences of thathexameric sequence pattern would not be expected in the regulatoryelements if the elements had the same hexamer composition as the entiregenome. Once the over-represented patterns are identified, a statisticaltest is applied to determine whether their over-representation issignificant, or may be due to chance. For this test, a significanceindex, “sig,” is calculated for each pattern. The significance index isderived from the probability of occurrence of each pattern, which isestimated by a binomial distribution. The probability takes into accountthe number of possible patterns (4096 for hexamers). The highest sigvalues correspond to the most overrepresented oligonucleotides (vanHelden et al., 1998). In practical terms, oligonucleotides with sig≧0are considered as over-represented. A pattern with sig≧0 is likely to beover-represented due to chance once (=10⁰) in the set of regulatoryelement sequences. However, at sig≧1 a pattern is expected to beover-represented once in ten (=10¹) sequence sets, sig≧2 once in 100(=10²) sequence sets, etc.

The patterns that are significantly over-represented in the regulatoryelement set are used to develop a model for classification andprediction of regulatory element sequences. This employs DiscriminantAnalysis, a so-called “supervised” method of statistical classificationknown to one of ordinary skill in the art (Huberty, 1994). InDiscriminant Analysis, sets of known or classified items (e.g., STARelements) are used to “train” a model to recognize those items on thebasis of specific variables (e.g., sequence patterns such as hexamers).The trained model is then used to predict whether other items should beclassified as belonging to the set of known items (e.g., is a DNAsequence STAR element). In this example, the known items in the trainingset are STAR elements (positive training set). They are contrasted withsequences that are randomly selected from the genome (negative trainingset) which have the same length as the STAR elements. DiscriminantAnalysis establishes criteria for discriminating positives fromnegatives based on a set of variables that distinguish the positives; inthis example, the variables are the significantly over-representedpatterns (e.g., hexamers).

When the number of over-represented patterns is high compared to thesize of the training set, the model could become biased due toover-training. Over-training is circumvented by applying a forwardstepwise selection of variables (Huberty, 1994). The goal of StepwiseDiscriminant Analysis is to select the minimum number of variables thatprovides maximum discrimination between the positives and negatives. Themodel is trained by evaluating variables one-by-one for their ability toproperly classify the items in the positive and negative training sets.This is done until addition of new variables to the model does notsignificantly increase the model's predictive power (i.e., until theclassification error rate is minimized). This optimized model is thenused for testing, in order to predict whether “new” items are positivesor negatives (Huberty, 1994).

It is inherent in classification statistics that for complex items suchas DNA sequences, some elements of the positive training set will beclassified as negatives (false negatives), and some members of thenegative training set will be classified as positives (false positives).When a trained model is applied to testing new items, the same types ofmisclassifications are expected to occur.

In the bio-informatic method described here, the first step, patternfrequency analysis, reduces a large set of sequence patterns (e.g., all4096 hexamers) to a smaller set of significantly over-representedpatterns (e.g., 100 hexamers); in the second step, Stepwise DiscriminantAnalysis reduces the set of over-represented patterns to the subset ofthose patterns that have maximal discriminative power (e.g., 5-10hexamers). Therefore, this approach provides simple and robust criteriafor identifying regulatory DNA elements such as STAR elements.

DNA-binding proteins can be distinguished on the basis of the type ofbinding site they occupy. Some recognize contiguous sequences; for thistype of protein, patterns that are oligonucleotides of length 6 basepairs (hexamers) are fruitful for bio-informatic analysis (van Helden etal., 1998). Other proteins bind to sequence dyads: contact is madebetween pairs of highly conserved trinucleotides separated by anon-conserved region of fixed width (van Helden et al., 2000). In orderto identify sequences in STAR elements that may be bound by dyad-bindingproteins, frequency analysis was also conducted for this type ofpattern, where the spacing between the two trinucleotides was variedfrom 0 to 20 (i.e., XXXN{0-20}XXX where X's are specific nucleotidescomposing the trinucleotides, and N's are random nucleotides from 0 to20 base pairs in length). The results of dyad frequency analysis arealso used for Linear Discriminant Analysis as described above.

Materials and Methods

Using the genetic screen described herein and in EP 01202581.3,sixty-six (66) STAR elements were initially isolated from human genomicDNA and characterized in detail (Table 6). The screen was performed ongene libraries constructed by Sau3AI digestion of human genomic DNA,either purified from placenta (Clontech 6550-1) or carried inbacterial/P1 (BAC/PAC) artificial chromosomes. The BAC/PAC clonescontain genomic DNA from regions of chromosome 1 (clones RP1154H19 andRP3328E19), from the HOX cluster of homeotic genes (clones RP1167F23,RP1170019, and RP11387A1), or from human chromosome 22 (ResearchGenetics 96010-22). The DNAs were size-fractionated, and the 0.5-2 kbsize fraction was ligated into BamHI-digested pSelect vector, bystandard techniques (Sambrook et al., 1989). pSelect plasmids containinghuman genomic DNA that conferred resistance to zeocin at low doxycyclineconcentrations were isolated and propagated in Escherichia coli. Thescreens that yielded the STAR elements of Table 6 have assayedapproximately 1-2% of the human genome.

The human genomic DNA inserts in these 66 plasmids were sequenced by thedideoxy method (Sanger et al., 1977) using a Beckman CEQ2000 automatedDNA sequencer, using the manufacturer's instructions. Briefly, DNA waspurified from E. coli using QIAprep® Spin Miniprep and Plasmid Midi Kits(QIAGEN® 27106 and 12145, respectively). Cycle sequencing was carriedout using custom oligonucleotides corresponding to the pSelect vector(primers D89 and D95, Table 1), in the presence of dye terminators (CEQ™Dye Terminator Cycle Sequencing Kit, Beckman 608000). Assembled STAR DNAsequences were located in the human genome (database builds August andDecember 2001) using BLAT (Basic Local Alignment Tool (Kent, 2002);WorldWideWeb.genome.ucsc.edu/cgi-bin/hgGateway; Table 6). In aggregate,the combined STAR sequences comprise 85.6 kilobase pairs, with anaverage length of 1.3 kilobase pairs.

Sequence motifs that distinguish STAR elements within human genomic DNAwere identified by bio-informatic analysis using a two-step procedure,as follows (see FIG. 20 for a schematic diagram). The analysis has twoinput datasets: (1) the DNA sequences of the STAR elements (STAR1-STAR65(SEQ ID NOS:1-65) were used; Table 6); and (2) the DNA sequence of thehuman genome (except for chromosome 1, which was not feasible to includedue to its large size; for dyad analysis a random subset of humangenomic DNA sequence (˜27 Mb) was used).

Pattern Frequency Analysis. The first step in the analysis usesRSA-Tools software (Regulatory Sequence Analysis Tools;WorldWideWeb.ucmb.ulb.ac.be/bioinformatics/rsa-tools/; references (vanHelden et al., 1998, van Helden et al., 2000, van Helden et al., 2000))to determine the following information: (1) the frequencies of all dyadsand hexameric oligonucleotides in the human genome; (2) the frequenciesof the oligonucleotides and dyads in the 65 STAR elements; and (3) thesignificance indices of those oligonucleotides and dyads that areover-represented in the STAR elements compared to the genome. A controlanalysis was done with 65 sequences that were selected at random fromthe human genome (i.e., from 2689×10³ kilobase pairs) that match thelength of the STAR elements of Table 6.

Discriminant Analysis. The over-represented oligonucleotides and dyadswere used to train models for prediction of STAR elements by LinearDiscriminant Analysis (Huberty, 1994). A pre-selection of variables wasperformed by selecting the 50 patterns with the highest individualdiscriminatory power from the over-represented oligos or dyads of thefrequency analyses. These pre-selected variables were then used formodel training in a Stepwise Linear Discriminant Analysis to select themost discriminant combination of variables (Huberty, 1994). Variableselection was based on minimizing the classification error rate(percentage of false negative classifications). In addition, theexpected error rate was estimated by applying the same discriminantapproach to the control set of random sequences (minimizing thepercentage of false positive classifications).

The predictive models from the training phase of Discriminant Analysiswere tested in two ways. First, the STAR elements and random sequencesthat were used to generate the model (the training sets) wereclassified. Second, sequences in a collection of 19 candidate STARelements (recently cloned by zeocin selection as described above) wereclassified. These candidate STAR elements are listed in Table 9 (SEQ IDNOS:67-84).

Results

Pattern frequency analysis was performed with RSA-Tools on 65 STARelements, using the human genome as the reference sequence. One hundredsixty-six (166) hexameric oligonucleotides were found to beover-represented in the set of STAR elements (sig≧0) compared to theentire genome (Table 4). The most significantly over-representedoligonucleotide, CCCCAC (SEQ ID NO:391), occurs 107 times among the 65STAR elements, but is expected to occur only 49 times. It has asignificance coefficient of 8.76; in other words, the probability thatits over-representation is due to random chance is 1/10^(8.76), i.e.,less than one in 500 million.

Ninety-five of the oligonucleotides have a significance coefficientgreater than one, and are, therefore, highly over-represented in theSTAR elements. Among the over-represented oligonucleotides, theirobserved and expected occurrences, respectively, range from six and one(for oligo 163, CGCGAA (SEQ ID NO:380), sig=0.02) to 133 and 95 (foroligo 120, CCCAGG (SEQ ID NO:337), sig=0.49). The differences inexpected occurrences reflect factors such as the G:C content of thehuman genome. Therefore, the differences among the oligonucleotides intheir number of occurrences is less important than theirover-representation; for example, oligo 2 (CAGCGG (SEQ ID NO:386)) is36/9=4-fold over-represented, which has a probability of being due torandom chance of one in fifty million (sig=7.75).

Table 4 also presents the number of STAR elements in which eachover-represented oligonucleotide is found. For example, the mostsignificant oligonucleotide, oligo 1 (CCCCAC (SEQ ID NO:391)), occurs107 times, but is found in only 51 STARs, i.e., on average it occurs astwo copies per STAR. The least abundant oligonucleotide, number 166(AATCGG (SEQ ID NO:383)), occurs on average as a single copy per STAR(thirteen occurrences on eleven STARs); single-copy oligonucleotidesoccur frequently, especially for the lower-abundance oligos. At theother extreme, oligo 4 (CAGCCC (SEQ ID NO:568)) occurs on average threetimes in those STARs in which it is found (37 STARs). The mostwidespread oligonucleotide is number 120 (CCCAGG (SEQ ID NO:337)), whichoccurs on 58 STARs (on average twice per STAR), and the least widespreadoligonucleotide is number 114 (CGTCGC (SEQ ID NO:331)), which occurs ononly 6 STARs (and on average only once per STAR).

Results of dyad frequency analysis are given in Table 5. Seven hundredthirty (730) dyads were found to be over-represented in the set of STARelements (sig≧0) compared to the reference sequence. The mostsignificantly over-represented dyad, CCCN{2}CGG (SEQ ID NO:384), occurs36 times among the 65 STAR elements, but is expected to occur only seventimes. It has a significance coefficient of 9.31; in other words, theprobability that its over-representation is due to chance is1/10^(9.31), i.e., less than one in two billion.

Three hundred ninety-seven of the dyads have a significance coefficientgreater than one, and are, therefore, highly over-represented in theSTAR elements. Among the over-represented dyads, their observed andexpected occurrences, respectively, range from nine and one (for fivedyads (numbers 380 (SEQ ID NO:763), 435 (SEQ ID NO:818), 493 (SEQ IDNO:876), 640 (SEQ ID NO:1023), and 665 (SEQ ID NO:1048))) to 118 and 63(for number 30 (AGGN{2}GGG) (SEQ ID NO:413), sig=4.44).

The oligonucleotides and dyads found to be over-represented in STARelements by pattern frequency analysis were tested for theirdiscriminative power by Linear Discriminant Analysis. Discriminantmodels were trained by step-wise selection of the best combination amongthe 50 most discriminant oligonucleotide (Table 4) or dyad (Table 5)patterns. The models achieved optimal error rates after incorporation offour (dyad) or five variables. The discriminative variables from oligoanalysis are numbers 11 (SEQ ID NO:228), 30 (SEQ ID NO:247), 94 (SEQ IDNO:311), 122 (SEQ ID NO:339), and 160 (SEQ ID NO:377) (Table 4); thosefrom dyad analysis are numbers 73 (SEQ ID NO:456), 194 (SEQ ID NO:577),419 (SEQ ID NO:802), and 497 (SEQ ID NO:880) (Table 5).

The discriminant models were then used to classify the 65 STAR elementsin the training set and their associated random sequences. The modelusing oligonucleotide variables classifies 46 of the 65 STAR elements asSTAR elements (true positives); the dyad model classifies 49 of the STARelements as true positives. In combination, the models classify 59 ofthe 65 STAR elements as STAR elements (91%; FIG. 21). The false positiverates (random sequences classified as STARs) were seven for the dyadmodel, eight for the oligonucleotide model, and 13 for the combinedpredictions of the two models (20%). The STAR elements of Table 6 thatwere not classified as STARs by LDA are STARs 7 (SEQ ID NO:7), 22 (SEQID NO:22), 35 (SEQ ID NO:35), 44 (SEQ ID NO:44), 46 (SEQ ID NO:46), and65 (SEQ ID NO:65). These elements display stabilizing anti-repressoractivity in functional assays, so the fact that they are not classifiedas STARs by LDA suggests that they represent another class (or classes)of STAR elements.

The models were then used to classify the 19 candidate STAR elements inthe testing set listed in Table 9. The dyad model classifies 12 of thesecandidate STARs as STAR elements, and the oligonucleotide modelclassifies 14 as STARs. The combined number of the candidates that areclassified as STAR elements is 15 (79%). This is a lower rate ofclassification than obtained with the training set of 65 STARs; this isexpected for two reasons. First, the discriminant models were trainedwith the 65 STARs of Table 6, and discriminative variables based on thistraining set may be less well represented in the testing set. Second,the candidate STAR sequences in the testing set have not yet been fullycharacterized in terms of in vivo function, and may include elementswith only weak anti-repression properties.

This analysis demonstrates the power of a statistical approach tobio-informatic classification of STAR elements. The STAR sequencescontain a number of dyad and hexameric oligonucleotide patterns that aresignificantly over-represented in comparison with the human genome as awhole. These patterns may represent binding sites for proteins thatconfer STAR activity; in any case they form a set of sequence motifsthat can be used to recognize STAR element sequences.

Using these patterns to recognize STAR elements by DiscriminantAnalysis, a high proportion of the elements obtained by the geneticscreen of the invention are in fact classified as STARs. This reflectsunderlying sequence and functional similarities among these elements. Animportant aspect of the method described here (pattern frequencyanalysis followed by Discriminant Analysis) is that it can bereiterated; for example, by including the 19 candidate STAR elements ofTable 9 with the 66 STAR elements of Table 6 into one training set, animproved discriminant model can be trained. This improved model can thenbe used to classify other candidate regulatory elements as STARs.Large-scale in vivo screening of genomic sequences using the method ofthe invention, combined with reiteration of the bio-informatic analysis,will provide a means of discriminating STAR elements that asymptoticallyapproaches 100% recognition and prediction of elements as the genome isscreened in its entirety. These stringent and comprehensive predictionsof STAR function will ensure that all human STAR elements arerecognized, and are available for use in improving transgene expression.

Example 14 Cloning and Characterization of STAR Elements fromArabidopsis thaliana

Transgene silencing occurs in transgenic plants at both thetranscriptional and post-transcriptional levels (Meyer, 2000, Vance &Vaucheret, 2001). In either case, the desired result of transgeneexpression can be compromised by silencing; the low expression andinstability of the transgene results in poor expression of desirabletraits (e.g., pest resistance) or low yields of recombinant proteins. Italso results in poor predictability: the proportion of transgenic plantsthat express the transgene at biotechnologically useful levels is low,which necessitates laborious and expensive screening of transformedindividuals for those with beneficial expression characteristics. Thisexample describes the isolation of STAR elements from the genome of thedicot plant Arabidopsis thaliana for use in preventing transcriptionaltransgene silencing in transgenic plants. Arabidopsis was chosen forthis example because it is a well-studied model organism: it has acompact genome, it is amenable to genetic and recombinant DNAmanipulations, and its genome has been sequenced (Bevan et al., 2001,Initiative, 2000, Meinke et al., 1998).

Materials and Methods:

Genomic DNA was isolated from Arabidopsis thaliana ecotype Columbia asdescribed (Stam et al., 1998) and partially digested with MboI. Thedigested DNA was size-fractionated to 0.5-2 kilobase pairs by agarosegel electrophoresis and purification from the gel (QIAquick® GelExtraction Kit, QIAGEN® 28706), followed by ligation into the pSelectvector (supra). Transfection into the U-2 OS/Tet-Off/LexA-HP1 cell lineand selection for zeocin resistance at low doxycycline concentration wasperformed as described (supra). Plasmids were isolated from zeocinresistant colonies and re-transfected into the U-2 OS/Tet-Off/LexA-HP1cell line.

Sequencing of Arabidopsis genomic DNA fragments that conferred zeocinresistance upon re-transfection was performed as described (supra). TheDNA sequences were compared to the sequence of the Arabidopsis genome byBLAST analysis ((Altschul et al., 1990); URLWorldWideWeb.ncbi.nlm.nih.gov/blast/Blast).

STAR activity was tested further by measuring mRNA levels for thehygromycin- and zeocin-resistance genes in recombinant host cells byreverse transcription PCR (RT-PCR). Cells of the U-2 OS/Tet-Off/lexA-HP1cell line were transfected with pSelect plasmids containing ArabidopsisSTAR elements, the Drosophila scs element, or containing no insert(supra). These were cultivated on hygromycin for two weeks at highdoxycycline concentration, then the doxycycline concentration waslowered to 0.1 ng/ml to induce the lexA-HP1 repressor protein. After tendays, total RNA was isolated by the RNeasy mini kit (QIAGEN® 74104) asdescribed by the manufacturer. First-strand cDNA synthesis was carriedout using the RevertAid™ First Strand cDNA Synthesis kit (MBI Fermentas1622) using oligo(dT)18 primer as described by the manufacturer. Analiquot of the cDNA was used as the template in a PCR reaction usingprimers D58 (SEQ ID NO:151) and D80 (SEQ ID NO:154) (for the zeocinmarker), and D70 (SEQ ID NO:152) and D71 (SEQ ID NO:153) (for thehygromycin marker), and Taq DNA polymerase (Promega M2661). The reactionconditions were 15-20 cycles of 94° C. for one minute, 54° C. for oneminute, and 72° C. for 90 seconds. These conditions result in a linearrelationship between input RNA and PCR product DNA. The PCR productswere resolved by agarose gel electrophoresis, and the zeocin andhygromycin bands were detected by Southern blotting as described(Sambrook et al., 1989), using PCR products produced as above withpurified pSelect plasmid as template. The ratio of the zeocin andhygromycin signals corresponds to the normalized expression level of thezeocin gene.

Results

The library of Arabidopsis genomic DNA in the pSelect vector comprised69,000 primary clones in E. coli, 80% of which carried inserts. Theaverage insert size was approximately 1000 base pairs; the library;therefore, represents approximately 40% of the Arabidopsis genome.

A portion of this library (representing approximately 16% of theArabidopsis genome) was transfected into the U-2 OS/Tet-Off/LexA-HP1cell line. Hygromycin selection was imposed to isolate transfectants,which resulted in 27,000 surviving colonies. These were then subjectedto zeocin selection at low doxycycline concentration. PutativeSTAR-containing plasmids from 56 zeocin-resistant colonies were rescuedinto E. coli and re-transfected into U-2 OS/Tet-Off/LexA-HP1 cells.Forty-four of these plasmids (79% of the plasmids tested) conferredzeocin resistance on the host cells at low doxycycline concentrations,demonstrating that the plasmids carried STAR elements. This indicatesthat the pSelect screen in human U-2 OS cells is highly efficient atdetection of STAR elements from plant genomic DNA.

The DNA sequences of these 44 candidate STAR elements were determined.Thirty-five of them were identified as single loci in the database ofArabidopsis nuclear genomic sequence (Table 10; SEQ ID NO:85-SEQ IDNO:119). Four others were identified as coming from the chloroplastgenome, four were chimeras of DNA fragments from two loci, and one wasnot found in the Arabidopsis genome database.

The strength of the cloned Arabidopsis STAR elements was tested byassessing their ability to prevent transcriptional repression of thezeocin-resistance gene, using an RT-PCR assay. As a control for RNAinput among the samples, the transcript levels of thehygromycin-resistance gene for each STAR transfection were assessed too.This analysis has been performed for 12 of the Arabidopsis STARelements. The results (FIG. 22) demonstrate that the Arabidopsis STARelements are superior to the Drosophila scs element (positive control)and the empty vector (“SV40”; negative control) in their ability toprotect the zeocin-resistance gene from transcriptional repression. Inparticular, STAR-A28 (SEQ ID NO:112) and STAR-A30 (SEQ ID NO:114) enable2-fold higher levels of zeocin-resistance gene expression than the scselement (normalized to the internal control of hygromycin-resistancegene mRNA) when the lexA-HP1 repressor is expressed.

These results demonstrate that the method of the invention can besuccessfully applied to recovery of STAR elements from genomes of otherspecies than human. Its successful application to STAR elements from aplant genome is particularly significant because it demonstrates thewide taxonomic range over which the method of the invention isapplicable, and because plants are an important target ofbiotechnological development.

Example 15 STAR-Shielded Genes That Reside on Multiple Vectors areExpressed Simultaneously in CHO Cells

STAR elements function to block the effect of transcriptional repressioninfluences on transgene expression units. One of the benefits of STARelements for heterologous protein production is the increasedpredictability of finding high-expressing primary recombinant hostcells. This feature allows for the simultaneous expression of differentgenes that reside on multiple, distinct vectors. In this example, we usetwo different STAR7-shielded (SEQ ID NO:7) genes, GFP and RED, which arelocated on two different vectors. When these two vectors are transfectedsimultaneously to Chinese hamster ovary (CHO) cells, both are expressed,whereas the corresponding, but unprotected GFP and RED genes, showhardly such simultaneous expression.

Material and Methods

The STAR7 element (SEQ ID NO:7) is tested in the ppGIZ-STAR7 andppRIP-STAR7 vectors (FIG. 23). The construction of the pPlug&Play (ppGIZand ppRIP) vectors is described below. Plasmid pGFP (Clontech 6010-1) ismodified by insertion of a linker at the BsiWI site to yield pGFP-link.The linker (made by annealing oligonucleotides5′GTACGGATATCAGATCTTTAATTAAG3′ (SEQ ID NO:124) and5′GTACCTTAATTAAAGATCTGATATCC3′ (SEQ ID NO:125)) introduces sites for thePacI, BglII, and EcoRV restriction endonucleases. This creates themultiple cloning site MCSII for insertion of STAR elements. Then primers(5′GATCAGATCTGGCGCGCCATTTAAATCG TCTCGCGCGTTTCGGTGATGACGG3′ (SEQ IDNO:126)) and (5′AGGCGGATCCGA ATGTATTTAGAAAAATAAACAAA TAGGGG3′ (SEQ IDNO:127)) are used to amplify a region of 0.37 kb from pGFP, which isinserted into the BglII site of pIRES (Clontech 6028-1) to yieldpIRES-stuf. This introduces sites for the AscI and SwaI restrictionendonucleases at MCSI, and acts as a “stuffer fragment” to avoidpotential interference between STAR elements and adjacent promoters.pIRES-stuf is digested with BglII and FspI to liberate a DNA fragmentcomposed of the stuffer fragment, the CMV promoter, the IRES element(flanked by multiple cloning sites MCS A and MCS B), and the SV40polyadenylation signal. This fragment is ligated with the vectorbackbone of pGFP-link produced by digestion with BamHI and StuI, toyield pIRES-link.

The open reading frames of the zeocin-resistance gene are inserted intothe BamHI/NotI sites of MCS B in pIRES-link as follows: thezeocin-resistance ORF is amplified by PCR with primers5′GATCGGATCCTTCGAAATGGCCAAGTTGACCAGTGC3′ (SEQ ID NO:128) and5′AGGCGCGGCCGCAATTCTCAGTCCTGCTCCTC3′ (SEQ ID NO:129) from plasmidpEM7/zeo, digested with BamHI and NotI, and ligated withBamHI/NotI-digested pIRES-link to yield pIRES-link-zeo. The GFP reporterORF is introduced into pIRES-link-zeo by amplification of phr-GFP-1 withprimers 5′GATCGAATTCTCGCGAATGGTGAGCAAGC AGATCCTGAAG3′ (SEQ ID NO:130)and 5′AGGCGAATTCACCGGTGTTTAAACTTAC ACCCACTCGTGCAGGCTGCCCAGG3′ (SEQ IDNO:131), and insertion of the EcoRI-digested GFP cassette into the EcoRIsite in MCS A of the pIRES-link-zeo plasmid. This creates the ppGIZ (forppGFP-IRES-zeo). 5′ STAR7 (SEQ ID NO:7) is cloned into the SalI site and3′ STAR7 (SEQ ID NO:7) is cloned into the PacI site.

The puromycin-resistance ORF is amplified by PCR with primers5′GATCGGATCCTTCGAAATGACCGAGTACAAGCCCACG3′ (SEQ ID NO:132) and5′AGGCGCGGCCGCTCAGGCACCGGGCTTGCGGGTC3′ (SEQ ID NO:133) from plasmidpBabe-Puro (Morgenstern & Land, 1990), digested with BamHI and NotI, andligated with BamHI/NotI-digested pIRES-link to yield pIRES-link-puro.The RED gene is amplified by PCR with primers5′GATCTCTAGATCGCGAATGGCCTCCTCCGAGAACGTCATC3′ (SEQ ID NO:134) and5′AGGCACGCGTTCGCGACTACAGGAACAGGTGGTGGCG3′ (SEQ ID NO:135) from plasmidpDsRed2 (Clontech 6943-1), digested with XbaI and MluI and ligated toNheI-MluI digested pIRES-link-puro to yield ppRIP (for ppRED-IRES-puro).5′ STAR7 (SEQ ID NO:7) is cloned into the SalI site and 3′ STAR7 (SEQ IDNO:7) is cloned into the PacI site.

Transfection and Culture of CHO Cells

The Chinese Hamster Ovary cell line CHO-K1 (ATCC CCL-61) is cultured inHAMS-F12 medium+10% Fetal Calf Serum containing 2 mM glutamine, 100 U/mlpenicillin, and 100 micrograms/ml streptomycin at 37° C./5% CO₂. Cellsare transfected with the plasmids using Lipofectamine™ 2000 (Invitrogen)as described by the manufacturer. Briefly, cells are seeded to culturevessels and grown overnight to 70-90% confluence. Lipofectamine reagentis combined with plasmid DNA at a ratio of 7.5 microliters per 3microgram (e.g., for a 10 cm Petri dish, 20 micrograms DNA and 120microliters Lipofectamine) and added after 30 minutes incubation at 25°C. to the cells. After six hours incubation, the transfection mixture isreplaced with fresh medium, and the transfected cells are incubatedfurther. After overnight cultivation, cells are trypsinized and seededinto fresh petri dishes with fresh medium with zeocin added to aconcentration of 100 μg/ml and the cells are cultured further. Whenindividual colonies become visible (approximately ten days aftertransfection) medium is removed and replaced with fresh medium(puromycin).

Individual colonies are isolated and transferred to 24-well plates inmedium with zeocin. Expression of the GFP and RED reporter genes isassessed approximately three weeks after transfection.

One tested construct consists of a monocistronic gene with the GFP gene,an IRES and the Zeocin resistance gene under control of the CMVpromoter, but either with or without STAR7 element (SEQ ID NO:7) toflank the entire construct (FIG. 23). The other construct consists of amonocistronic gene with the RED gene, an IRES and the puromycinresistance gene under control of the CMV promoter, but either with orwithout STAR7 element (SEQ ID NO:7) to flank the entire construct (FIG.23).

The constructs are transfected to CHO-K1 cells. Stable colonies that areresistant for both zeocin and puromycin are expanded before the GFP andRED signals are determined on a XL-MCL Beckman Coulter flow cytometer.The percentage of cells in one colony that are double positive for bothGFP and RED signals is taken as measure for simultaneous expression ofboth proteins and this is plotted in FIG. 23.

Results

FIG. 23 shows that simultaneous expression in independent zeocin andpuromycin resistant CHO colonies of GFP and a RED reporter genes thatare flanked by a STAR element results in a higher number of cells thatexpress both GFP and RED proteins, as compared to the control vectorswithout STAR7 element (SEQ ID NO:7). The STAR7 element (SEQ ID NO:7),therefore, conveys a higher degree of predictability of transgeneexpression in CHO cells. In the STAR-less colonies at most nine out of20 colonies contain double GFP/RED positive cells. The percentage ofdouble positive cells ranges between 10 and 40%. The remaining 11 out of20 colonies have less than 10% GFP/RED positive cells. In contrast, in19 out of 20 colonies that contain the STAR-shielded GFP and RED genes,the percentage GFP/RED double positive cells ranges between 25 and 75%.In 15 out of these 19 double positive colonies the percentage GFP/REDdouble positive cells is higher than 40%. This result shows that it ismore likely that simultaneous expression of two genes is achieved whenthese genes are flanked with STAR elements.

Example 16 Expression of a Functional Antibody From Two SeparatePlasmids is More Easily Obtained When STAR Elements Flank the GenesEncoding the Heavy and Light Chains

Due to the ability of STAR elements to convey higher predictability toprotein expression two genes can be expressed simultaneously fromdistinct vectors. This is shown in Example 15 for two reporter genes,GFP and RED. Now the simultaneous expression of a light and a heavyantibody chain is tested. In Example 16, STAR7-shielded light and heavyantibody cDNAs that reside on distinct vectors are simultaneouslytransfected to Chinese hamster ovary cells. This results in theproduction of functional antibody, indicating that both heavy and lightchains are expressed simultaneously. In contrast, the simultaneoustransfection of unprotected light and heavy antibody cDNAs shows hardlyany expression of functional antibody.

Materials and Methods

The tested constructs are the same as described in Example 15, exceptthat the GFP gene is replaced by the gene encoding the light chain ofthe RING1 antibody (Hamer et al., 2002) and the RED gene is replaced bythe gene encoding the heavy chain of the RING1 antibody. The light chainis amplified from the RING1 hybridoma (Hamer et al., 2002) by RT-PCRusing the primers 5′CAAGAATTCAATGGATTTTCAAGTGCAG3′ (SEQ ID NO:136) and5′CAAGCGGCCGCTTTGTCTCTAACACTCATTCC3′ (SEQ ID NO:137). The PCR product iscloned into pcDNA3 after restriction digestion with EcoRI and NotI andsequenced to detect potential frame shifts in the sequence. The cDNA isexcised with EcoRI and NotI, blunted and cloned in ppGIZ plasmid. Theheavy chain is amplified from the RING1 hybridoma (Hamer et al., 2002)by RT-PCR using the primers 5′ACAGAATTCTTACCATGGATTTTGGGCTG3′ (SEQ IDNO:138) and 5′ACAGCGGCCGCTCATTTACCAGGAGAGTGGG3′ (SEQ ID NO:139). The PCRproduct is cloned into pcDNA3 after restriction digestion with EcoRI andNotI and sequenced to detect potential frame shifts in the sequence. ThecDNA is excised with EcoRI and NotI, blunted and cloned in ppRIPplasmid.

Results

CHO colonies are simultaneously transfected with the RING1 Light Chain(LC) and RING1 Heavy Chain (HC) cDNAs that reside on two distinctvectors. The Light Chain is coupled to the zeocin resistance genethrough an IRES, the Heavy Chain is coupled to the puromycin resistancegene through an IRES. FIG. 24 shows that simultaneous transfection toCHO cells of the heavy and light chain encoding cDNAs results in theestablishment of independent zeocin and puromycin resistant colonies.When the constructs are flanked by the STAR7 element (SEQ ID NO:7), thisresults in a higher production of functional RING1 antibody, as comparedto the control vectors without STAR7 element (SEQ ID NO:7). The STAR7element (SEQ ID NO:7), therefore, conveys a higher degree ofpredictability of antibody expression in CHO cells.

In the STAR-less colonies only one out of 12 colonies express detectableantibody. In contrast, seven out of twelve colonies that contain theSTAR-shielded Light and Heavy Chain genes produce functional RING1antibody that detects the RING1 antigen in an ELISA assay.Significantly, all these seven colonies produce higher levels of RING1antibody than the highest control colony (arbitrarily set at 100%). Thisresult shows that it is more likely that simultaneous expression of twogenes encoding two antibody chains is achieved when these genes areflanked with STAR elements.

TABLE 1 Oligonucleotides used for polymerase chain reactions (PCRprimers) or DNA mutagenesis (SEQ ID NOS: 140-207). SEQ ID Num- NO: berSequence 140 C65 AACAAGCTTGATATCAGATCTGCTAGCTTGGTCGAGCTGATA CTTCCC 141C66 AAACTCGAGCGGCCGCGAATTCGTCGACTTTACCACTCCCTA TCAGTGATAGAG 142 C67AAACCGCGGCATGGAAGACGCCAAAAACATAAAGAAAGG 143 C68TATGGATCCTAGAATTACACGGCGATCTTTCC 144 C81AAACCATGGCCGAGTACAAGCCCACGGTGCGCC 145 C82AAATCTAGATCAGGCACCGGGCTTGCGGGTCATGC 146 C85 CATTTCCCCGAAAAGTGCCACC 147D30 TCACTGCTAGCGAGTGGTAAACTC 148 D41 GAAGTCGACGAGGCAGGCAGAAGTATGC 149D42 GAGCCGCGGTTTAGTTCCTCACCTTGTCG 150 D51 TCTGGAAGCTTTGCTGAAGAAAC 151D58 CCAAGTTGACCAGTGCC 152 D70 TACAAGCCAACCACGGCCT 153 D71CGGAAGTGCTTGACATTGGG 154 D80 GTTCGTGGACACGACCTCCG 155 D89GGGCAAGATGTCGTAGTCAGG 156 D90 AGGCCCATGGTCACCTCCATCGCTACTGTG 157 D91CTAATCACTCACTGTGTAAT 158 D93 AATTACAGGCGCGCC 159 D94 AATTGGCGCGCCTGT 160D95 TGCTTTGCATACTTCTGCCTGCCTC 161 E12 TAGGGGGGATCCAAATGTTC 162 E13CCTAAAAGAAGATCTTTAGC 163 E14 AAGTGTTGGATCCACTTTGG 164 E15TTTGAAGATCTACCAAATGG 165 E16 GTTCGGGATCCACCTGGCCG 166 E17TAGGCAAGATCTTGGCCCTC 167 E18 CCTCTCTAGGGATCCGACCC 168 E19CTAGAGAGATCTTCCAGTAT 169 E20 AGAGTTCCGGATCCGCCTGG 170 E21CCAGGCAGACTCGGAACTCT 171 E22 TGGTGAAACCGGATCCCTAC 172 E23AGGTCAGGAGATCTAGACCA 173 E25 CCATTTTCGCTTCCTTAGCTCC 174 E42CGATGTAACCCACTCGTGCACC 175 E57 AGAGATCTAGGATAATTTCG 176 E84GATCTCTAGAATGGCCAAGCCTTTGTCTCAAG 177 E85AGGCGCGGCCGCTTAGCCCTCCCACACATAACCAGAG 178 E87AGGCACGCGTTCATGTCTGCTCGAAGCGGCC 179 E92AGGCGCTAGCACGCGTTCTACTCTTTTCCTACTCTG 180 E93GATCAAGCTTACGCGTCTAAAGGCATTTTATATAG 181 E94AGGCGCTAGCACGCGTTCAGAGTTAGTGATCCAGG 182 E95GATCAAGCTTACGCGTCAGTAAAGGTTTCGTATGG 183 E96AGGCGCTAGCACGCGTTCTACTCTTTCATTACTCTG 184 E97 CGAGGAAGCTGGAGAAGGAGAAGCTG185 E98 CAAGGGCCGCAGCTTACACATGTTC 186 E99 GATCACTAGTATGGCCAAGTTGACCAGTGC187 E100 AGGCGCGGCCGCAATTCTCAGTCCTGCTCCTC 188 F11GATCGCTAGCAATCGCGACTTCGCCCACCATGC 189 F14GATCGAATTCTCGCGACTTCGCCCACCATGC 190 F15AGGCGAATTCACCGGTGTTTAAACTCATGTCTGCTCGAAGCG GCCGG 191 F16GATCGAATTCTCGCGAATGGTGAGCAAGCAGATCCTGAAG 192 F17AGGCGAATTCACCGGTGTTTAAACTTACACCCACTCGTGCAG GCTGCCCAGG 193 F18GATCGGATCCTTCGAAATGGCCAAGTTGACCAGTGC 194 F19GATCGGATCCTTCGAAATGATTGAACAAGATGGATTGC 195 F20AGGCGCGGCCGCTCAGAAGAACTCGTCAAGAAGGCG 196 F21GATCGGATCCTTCGAAATGACCGAGTACAAGCCCACG 197 F22AGGCGCGGCCGCTCAGGCACCGGGCTTGCGGGTC 198 F23GATCAGATCTGGCGCGCCATTTAAATCGTCTCGCGCGTTTCG GTGATGACGG 199 F24AGGCGGATCCGAATGTATTTAGAAAAATAAACAAATAGGGG 200 F25GTACGGATATCAGATCTTTAATTAAG 201 F26 GTACCTTAATTAAAGATCTGATATCC 202 F32GATCGAGGTACCGGTGTGT 203 F33 GATCACACACCGGTACCTC 204 F34CGGAGGTACCGGTGTGT 205 F35 CGACACACCGGTACCTC 206 F44 TGAGAGGTACCGGTGTGT207 F45 TCAACACACCGGTACCTC

TABLE 2 STAR elements and two-step selection increase the predictabilityof transgene expression fold without STAR improvement with STAR (carryout first antibiotic selection) Number colonies¹ ~100 10-fold ~1000 Highproducers percent 5%  3-fold 15% number 5 150 (characterize 20 (20% ofpopulation) (2% colonies) of population) High producers 1  3-fold² 3 Lowproducers 19 17 (carry out second antibiotic selection, killing lowproducers) Survivors to 5 30-fold³ 150 characterize ¹Colonies permicrogram plasmid DNA. ²Manifesting the three-fold improvement due tothe presence of STARs in the percent of high producers in the originalpopulation of colonies resistant to the first antibiotic. ³Manifestingthe arithmetic product of the fold improvement in the number of coloniesand the increased percentage of high producers due to the presence ofSTARs.

TABLE 3 Sequences of various STAR elements (SEQ ID NOS: 208-217) STAR3forward (SEQ ID NO: 208)ACGTNCTAAGNAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCACTCGAGCGGCCAGCTTGGATCTCGAGTACTGAAATAGGAGTAAATCTGAAGAGCAAATAAGATGAGCCAGAAAACCATGAAAAGAACAGGGACTACCAGTTGATTCCACAAGGACATTCCCAAGGTGAGAAGGCCATATACCTCCACTACCTGAACCAATTCTCTGTATGCAGATTTAGCAAGGTTATAAGGTAGCAAAAGATTAGACCCAAGAAAATAGAGAACTTCCAATCCAGTAAAAATCATAGCAAATTTATTGATGATAACAATTGTCTCCAAAGGAACCAGGCAGAGTCGTGCTAGCAGAGGAAGCACGTGAGCTGAAAACAGCCAAATCTGCTTTGTTTTCATGACACAGGAGCATAAAGTACACACCACCAACTGACCTATTAAGGCTGTGGTAAACCGATTCATAGAGAGAGGTTCTAAATACATTGGTCCCTCATAGGCAAACCGCAGTTCACTCCGAACGTAGTCCCTGGAAATTTGATGTCCAGNATAGAAAAGCANAGCAGNCNNNNNNTATANATNNNGNTGANCCANATGNTNNCTGNNC STAR3 reverse (SEQ ID NO:209) GAGCTAGCGGCGCGCCAAGCTTGGATCCCGCCCCGCCCCCTCCGCCCTCGAGCCCCGCCCCTTGCCCTAGAGGCCCTGCCGAGGGGCGGGGCCTGTCCCTCCTCCCCTTTCCCCCGCCCCCTACCGTCACGCTCAGGGGCAGCCTGACCCCGAGCGGCCCCGCGGTGACCCTCGCGCAGAGGCCTGTGGGAGGGGCGTCGCAAGCCCCTGAATCCCCCCCCGTCTGTTCCCCCCTCCCGCCCAGTCTCCTCCCCCTGGGAACGCGCGGGGTGGGTGACAGACCTGGCTGCGCGCCACCGCCACCGCGCCTGCCGGGGGCGCTGCCGCTGCCTGAGAAACTGCGGCTGCCGCCTGGAGGAGGTGCCGTCGCCTCCGCCACCGCTGCCGCCGCCGCCAGGGGTAGGAGCTAAGCCGCCGCCATTTTGTGTCCCCCTGTTGTTGTCGTTGACATGAATCCGACATGACACTGATTACAGCCCAATGGAGTCTCATTAAACCCGAGTCGCGGTCCCGCCCCGCCGCTGCTCCATTGGAGGAGACCAAAGACACTTAAGGCCACCCGTTGGCCTACGGGTCTGTCTGTCACCCACTCACTAACCACTCTGCAGCCCATTGGGGCAGGTTCCTGCCGGTCATNTCGCTTCCAATAAACACACCCCTTCGACCCCATNATTCCCCCCCTTCGGGAACCACCCCCGGGGGAGGGGTCCACTGGNCAATACCAATTNAANAGAACCGCTNGGGTCCGCCTNTTTNCGGGCNCCCTAT TGGGTT STAR4forward (SEQ ID NO: 210)GGGGAGGATTCTTTTGGCTGCTGAGTTGAGATTAGGTTGAGGGTAGTGAAGGTAAAGGCAGTGAGACCACGTAGGGGTCATTGCAGTAATCCAGGCTGGAGATGATGGTGGTTCAGTTGGAATAGCAGTGCATGTGCTGTAACAACCTCAGCTGGGAAGCAGTATATGTGGCGTTATGACCTCAGCTGGAACAGCAATGCATGTGGTGGTGTAATGACCCCAGCTGGGTAGGGTGCATGTGATGGAACAACCTCAGCTGGGTAGCAGTGTACTTGATAAAATGTTGGCATACTCTACATTTGTTATGAGGGTAGTGCCATTAAATTTCTCCACAAATTGGTTGTCACGTATGAGTGAAAAGAGGAAGTGATGGAAGACTTCAGTGCTTTTGGCCTGAATAAATAGAAGACGTCATTTTCAGTAATGGAGACAGGGAAGACTAANGNAGGGTGGATTCAGTAGAGCAGGTGTTCAGTTTTGAATATGATGAACTCTGAGAGAGGAAAAACTTTTTCTACCTCTTAGTTTTTGNGNCTGGACTTAANATTAAAGGACATANGACNGAGANCAGACCAAATNTGCGANGTTTTTATATTTTACTTGCNGAGGGAATTTNCAAGAAAAAGAAGACCCAANANCCATTGGTCAAAACTATNTGCCTTTTAANAAAAAGANAATTACAATGGANANANAAGTGTTGNCTNG GCAAAAATTGGGSTAR4 reverse (SEQ ID NO: 211)GGATTNGAGCTAGCGGCGCGCCAAGCTTGGATCTTAGAAGGACAGAGTGGGGCATGGAAATGCACCACCAGGGCAGTGCAGCTTGGTCACTGCCAGCTCCNCTCATGGGCAGAGGGCTGGCCTCTTGCAGCCGACCAGGCACTGAGCGCCATCCCAGGGCCCTCGCCAGCCCTCAGCAGGGCCAGGACACACAAGCCTTTGACTTCCTCCTGTCACTGCTGCTGCCATTCCTGTTTTGTGGTCATCACTCCTTCCCTGTCCTCAGACTGCCCAGCACTCAAGGATGTCCTGTGGTGGCATCAGACCATATGCCCCTGAANAGGAGTGAGTTGGTGTTTTTTGCCGCGCCCANAGAGCTGCTGTCCCCTGAAAGATGCAAGTGGGAATGATGATGNTCACCATCNTCTGACACCAAGCCCTTTGGATAGAGGCCCCAACAGTGAGGATGGGGCTGCACTGCATTGCCAAGGCAACTCTGTNNTGACTGCTACANGACANTCCCAGGACCTGNGAAGNNCTATANATNTGAT GCNAGGCACCTSTAR6 forward (SEQ ID NO: 212)CCACCACAGACATCCCCTCTGGCCTCCTGAGTGGTTTCTTCAGCACAGCTTCCAGAGCCAAATTAAACGTTCACTCTATGTCTATAGACAAAAAGGGTTTTGACTAAACTCTGTGTTTTAGAGAGGGAGTTAAATGCTGTTAACTTTTTAGGGGTGGGCGAGAGGAATGACAAATAACAACTTGTCTGAATGTTTTACATTTCTCCCCACTGCCTCAAGAAGGTTCACAACGAGGTCATCCATGATAAGGAGTAAGACCTCCCAGCCGGACTGTCCCTCGGCCCCCAGAGGACACTCCACAGAGATATGCTAACTGGACTTGGAGACTGGCTCACACTCCAGAGAAAAGCATGGAGCACGAGCGCACAGAGCANGGGCCAAGGTCCCAGGGACNGAATGTCTAGGAGGGAGATTGGGGTGAGGGTANTCTGATGCAATTACTGNGCAGCTCAACATTCAAGGGAGGGGAAGAAAGAAACNGTCCCTGTAAGTAAGTTGTNCANCAGAGATGGTAAGCTCCAAATTTNAACTTTGGCTGCTGGAAAGTTTNNGGGCCNANANAANAAACANAAANATTTGAGGTTTANACCCACTAACCCNTATNANTANTTATTAATACCCCTAATTANACCTTGGATANCCTTAAAATATCNTNTNAAACGGAACCCTCNTTCCCNTTTNNAAATNNNAAAGGCCATTNNGNNCNAGTAAAAATCTNNNTTAAGNNNTGGGCCCNAACAAACNTNTTCCNAGACACNTTTTTTNTCCNGGNATTTNTAATTTATTTCTAANCC STAR6 reverse (SEQ ID NO: 213)ATCGTGTCCTTTCCAGGGACATGGATGAAGCTGGAAGCCATCATCCTCAGCAAACTAACACAGGAACAGAAAACCAAATACCACATGTTCTCACTCATAAGTGGGAGCTGAACAGTGAGAACACATGGACACAGGGAGGGGAACATCACACACCAAGGCCTGTCTGGTGTGGGGAGGGGAGGGAGAGCATCAGGACAAATAGCTAATGCATGTGGGGCTTAAACCTAGATGACGGGTTGATAGGTGCAGCAATCCACTATGGACACATATACCTATGTAACAACCCNACCTTNTTGACATGTATCCCAGAACTTAAAGGAAAATAAAAATTAAAAAAAATTNCCCTGGAATAAAAAAGAGTGTGGACTTTGGTGAGATN STAR8 forward (SEQ ID NO: 214)GGATCACCTCGAAGAGAGTCTAACGTCCGTAGGAACGCTCTCGGGTTCACAAGGATTGACCGAACCCCAGGATACGTCGCTCTCCATCTGAGGCTTGNTCCAAATGGCCCTCCACTATTCCAGGCACGTGGGTGTCTCCCCTAACTCTCCCTGCTCTCCTGAGCCCATGCTGCCTATCACCCATCGGTGCAGGTCCTTTCTGAANAGCTCGGGTGGATTCTCTCCATCCCACTTCCTTTCCCAAGAAAGAAGCCACCGTTCCAAGACACCCAATGGGACATTCCCNTTCCACCTCCTTNTCNAAAGTTNGCCCAGGTGTTCNTAACAGGTTAGGGAGAGAANCCCCCAGGTTTNAGTTNCAAGGCATAGGACGCTGGCTTGAACACACACACACNCTC STAR8 reverse (SEQ ID NO:215) GGATCCCGACTCTGCACCGCAAACTCTACGGCGCCCTGCAGGACGGCGGCCTCCTGCCGCTTGGACGCCAGNCAGGAGCTCCCCGGCAGCAGCAGAGCAGAAAGAAGGATGGCCCCGCCCCACTTCGCCTCCCGGCGGTCTCCCTCCCGCCGGCTCACGGACATAGATGGCTGCCTAGCTCCGGAAGCCTAGCTCTTGTTCCGGGCATCCTAAGGAAGACACGGTTTTTCCTCCCGGGGCCTCACCACATCTGGGACTTTGACGACTCGGACCTCTCTCCATTGAATGGTTGCGCGTTCTCTGGGAAAG STAR18 forward (SEQ ID NO: 216)TGGATCCTGCCGCTCGCGTCTTAGTGTTTCTCCCTCAAGACTTTCCTTCTGTTTTGTTGTCTTGTGCAGTATTTTACAGCCCCTCTTGTGTTTTTCTTTATTTCTCGTACACACACGCAGTTTTAAGGGTGATGTGTGTATAATTAAAAGGACCCTTGGCCCATACTTTCCTAATTCTTTAGGGACTGGGATTGGGTTTGACTGAAATATGTTTTGGTGGGGATGGGACGGTGGACTTCCATTCTCCCTAAACTGGAGTTTTGGTCGGTAATCAAAACTAAAAGAAACCTCTGGGAGACTGGAAACCTGATTGGAGCACTGAGGAACAAGGGAATGAAAAGGCAGACTCTCTGAACGTTTGATGAAATGGACTCTTGTGAAAATTAACAGTGAATATTCACTGTTGCACTGTACGAAGTCTCTGAAATGTAATTAAAAGTTTTTATTGAGCCCCCGAGCTTTGGCTTGCGCGTATTTTTCCGGTCGCGGACATCCCACCGCGCAGAGCCTCGCCTCCCCGCTGNCCTCAGCTCCGATGACTTCCCCGCCCCCGCCCTGCTCGGTGACAGACGTTCTACTGCTTCCAATCGGAGGC ACCCTTCGCGGSTAR18 reverse (SEQ ID NO: 217)TGGATCCTGCCGCTCGCGTCTTAGTGTTTCTCCCTCAAGACTTTCCTTCTGTTTTGTTGTCTTGTGCAGTATTTTACAGCCCCTCTTGTGTTTTTCTTTATTTCTCGTACACACACGCAGTTTTAAGGGTGATGTGTGTATAATTAAAAGGACCCTTGGCCCATACTTTCCTAATTCTTTAGGGACTGGGATTGGGTTTGACTGAAATATGTTTTGGTGGGGATGGGACGGTGGACTTCCATTCTCCCTAAACTGGAGTTTTGGTCGGTAATCAAAACTAAAAGAAACCTCTGGGAGACTGGAAACCTGATTGGAGCACTGAGGAACAAGGGAATGAAAAGGCAGACTCTCTGAACGTTTGATGAAATGGACTCTTGTGAAAATTAACAGTGAATATTCACTGTTGCACTGTACGAAGTCTCTGAAATGTAATTAAAAGTTTTTATTGAGCCCCCGAGCTTTGGC

The patterns are ranked according to significance coefficient. Thesewere determined using RSA-Tools with the sequence of the human genome asreference. Patterns that comprise the most discriminant variables inLinear Discriminant Analysis are indicated with an asterisk (SEQ IDNOS:218-383).

TABLE 4 Oligonucleotide patterns (6 base pairs) over- represented inSTAR elements Signifi- Number Oligonu- Observed Expected cance of SEQcleotide occur- occur- coef- matching ID Number sequence rences rencesficient STARs NO: 1 CCCCAC 107 49 8.76 51 218 2 CAGCGG 36 9 7.75 23 2193 GGCCCC 74 31 7.21 34 220 4 CAGCCC 103 50 7.18 37 221 5 GCCCCC 70 296.97 34 222 6 CGGGGC 40 12 6.95 18 223 7 CCCCGC 43 13 6.79 22 224 8CGGCAG 35 9 6.64 18 225 9 AGCCCC 83 38 6.54 40 226 10 CCAGGG 107 54 6.5243 227 11 GGACCC * 58 23 6.04 35 228 12 GCGGAC 20 3 5.94 14 229 13CCAGCG 34 10 5.9 24 230 14 GCAGCC 92 45 5.84 43 231 15 CCGGCA 28 7 5.6116 232 16 AGCGGC 27 7 5.45 17 233 17 CAGGGG 86 43 5.09 43 234 18 CCGCCC43 15 5.02 18 235 19 CCCCCG 35 11 4.91 20 236 20 GCCGCC 34 10 4.88 18237 21 GCCGGC 22 5 4.7 16 238 22 CGGACC 19 4 4.68 14 239 23 CGCCCC 35 114.64 19 240 24 CGCCAG 28 8 4.31 19 241 25 CGCAGC 29 8 4.29 20 242 26CAGCCG 32 10 4 24 243 27 CCCACG 33 11 3.97 26 244 28 GCTGCC 78 40 3.9 43245 29 CCCTCC 106 60 3.87 48 246 30 CCCTGC * 92 50 3.83 42 247 31 CACCCC77 40 3.75 40 248 32 GCGCCA 30 10 3.58 23 249 33 AGGGGC 70 35 3.55 34250 34 GAGGGC 66 32 3.5 40 251 35 GCGAAC 14 2 3.37 13 252 36 CCGGCG 17 43.33 12 253 37 AGCCGG 34 12 3.29 25 254 38 GGAGCC 67 34 3.27 40 255 39CCCCAG 103 60 3.23 51 256 40 CCGCTC 24 7 3.19 19 257 41 CCCCTC 81 443.19 43 258 42 CACCGC 33 12 3.14 22 259 43 CTGCCC 96 55 3.01 42 260 44GGGCCA 68 35 2.99 39 261 45 CGCTGC 28 9 2.88 22 262 46 CAGCGC 25 8 2.7719 263 47 CGGCCC 28 10 2.73 19 264 48 CCGCCG 19 5 2.56 9 265 49 CCCCGG30 11 2.41 17 266 50 AGCCGC 23 7 2.34 17 267 51 GCACCC 55 27 2.31 38 26852 AGGACC 54 27 2.22 33 269 53 AGGGCG 24 8 2.2 18 270 54 CAGGGC 81 472.18 42 271 55 CCCGCC 45 21 2.15 20 272 56 GCCAGC 66 36 2.09 39 273 57AGCGCC 21 6 2.09 18 274 58 AGGCCC 64 34 2.08 32 275 59 CCCACC 101 622.05 54 276 60 CGCTCA 21 6 2.03 17 277 61 AACGCG 9 1 1.96 9 278 62GCGGCA 21 7 1.92 14 279 63 AGGTCC 49 24 1.87 36 280 64 CCGTCA 19 6 1.7814 281 65 CAGAGG 107 68 1.77 47 282 66 CCCGAG 33 14 1.77 22 283 67CCGAGG 36 16 1.76 25 284 68 CGCGGA 11 2 1.75 8 285 69 CCACCC 87 53 1.7145 286 70 CCTCGC 23 8 1.71 20 287 71 CAAGCC 59 32 1.69 40 288 72 TCCGCA18 5 1.68 17 289 73 CGCCGC 18 5 1.67 9 290 74 GGGAAC 55 29 1.63 39 29175 CCAGAG 93 58 1.57 49 292 76 CGTTCC 19 6 1.53 16 293 77 CGAGGA 23 81.5 19 294 78 GGGACC 48 24 1.48 31 295 79 CCGCGA 10 2 1.48 8 296 80CCTGCG 24 9 1.45 17 297 81 CTGCGC 23 8 1.32 14 298 82 GACCCC 47 24 1.3133 299 83 GCTCCA 66 38 1.25 39 300 84 CGCCAC 33 15 1.19 21 301 85 GCGGGA23 9 1.17 18 302 86 CTGCGA 18 6 1.15 15 303 87 CTGCTC 80 49 1.14 50 30488 CAGACG 23 9 1.13 19 305 89 CGAGAG 21 8 1.09 17 306 90 CGGTGC 18 61.06 16 307 91 CTCCCC 84 53 1.05 47 308 92 GCGGCC 22 8 1.04 14 309 93CGGCGC 14 4 1.04 13 310 94 AAGCCC * 60 34 1.03 42 311 95 CCGCAG 24 91.03 17 312 96 GCCCAC 59 34 0.95 35 313 97 CACCCA 92 60 0.93 49 314 98GCGCCC 27 11 0.93 18 315 99 ACCGGC 15 4 0.92 13 316 100 CTCGCA 16 5 0.8914 317 101 ACGCTC 16 5 0.88 12 318 102 CTGGAC 58 33 0.88 32 319 103GCCCCA 67 40 0.87 38 320 104 ACCGTC 15 4 0.86 11 321 105 CCCTCG 21 8 0.818 322 106 AGCCCG 22 8 0.79 14 323 107 ACCCGA 16 5 0.78 13 324 108AGCAGC 79 50 0.75 41 325 109 ACCGCG 14 4 0.69 7 326 110 CGAGGC 29 130.69 24 327 111 AGCTGC 70 43 0.64 36 328 112 GGGGAC 49 27 0.64 34 329113 CCGCAA 16 5 0.64 12 330 114 CGTCGC 8 1 0.62 6 331 115 CGTGAC 17 60.57 15 332 116 CGCCCA 33 16 0.56 22 333 117 CTCTGC 97 65 0.54 47 334118 AGCGGG 21 8 0.52 17 335 119 ACCGCT 15 5 0.5 11 336 120 CCCAGG 133 950.49 58 337 121 CCCTCA 71 45 0.49 39 338 122 CCCCCA * 77 49 0.49 42 339123 GGCGAA 16 5 0.48 14 340 124 CGGCTC 29 13 0.47 19 341 125 CTCGCC 20 80.46 17 342 126 CGGAGA 20 8 0.45 14 343 127 TCCCCA 95 64 0.43 52 344 128GACACC 44 24 0.42 33 345 129 CTCCGA 17 6 0.42 13 346 130 CTCGTC 17 60.42 14 347 131 CGACCA 13 4 0.39 11 348 132 ATGACG 17 6 0.37 12 349 133CCATCG 17 6 0.37 13 350 134 AGGGGA 78 51 0.36 44 351 135 GCTGCA 77 500.35 43 352 136 ACCCCA 76 49 0.33 40 353 137 CGGAGC 21 9 0.33 16 354 138CCTCCG 28 13 0.32 19 355 139 CGGGAC 16 6 0.3 10 356 140 CCTGGA 88 59 0.345 357 141 AGGCGA 18 7 0.29 17 358 142 ACCCCT 54 32 0.28 36 359 143GCTCCC 56 34 0.27 36 360 144 CGTCAC 16 6 0.27 15 361 145 AGCGCA 16 60.26 11 362 146 GAAGCC 62 38 0.25 39 363 147 GAGGCC 79 52 0.22 42 364148 ACCCTC 54 32 0.22 33 365 149 CCCGGC 37 20 0.21 21 366 150 CGAGAA 208 0.2 17 367 151 CCACCG 29 14 0.18 20 368 152 ACTTCG 16 6 0.17 14 369153 GATGAC 48 28 0.17 35 370 154 ACGAGG 23 10 0.16 18 371 155 CCGGAG 208 0.15 18 372 156 ACCCAC 60 37 0.12 41 373 157 CTGGGC 105 74 0.11 50 374158 CCACGG 23 10 0.09 19 375 159 CGGTCC 13 4 0.09 12 376 160 AGCACC * 5433 0.09 40 377 161 ACACCC 53 32 0.08 38 378 162 AGGGCC 54 33 0.08 30 379163 CGCGAA 6 1 0.02 6 380 164 GAGCCC 58 36 0.02 36 381 165 CTGAGC 71 460.02 45 382 166 AATCGG 13 4 0.02 11 383

The patterns are ranked according to significance coefficient. Thesewere determined using RSA-Tools with the random sequence from the humangenome as reference. Patterns that comprise the most discriminantvariables in Linear Discriminant Analysis are indicated with an asterisk(SEQ ID NOS:384-1113).

TABLE 5 Dyad patterns over-represented in STAR elements. ObservedExpected Signifi- SEQ Num- Dyad occur- occur- cance ID ber sequencerences rences coefficient NO: 1 CCCN{2}CGG 36 7 9.31 384 2 CCGN{6}CCC 4010 7.3 385 3 CAGN{0}CGG 36 8 7.13 386 4 CGCN{15}CCC 34 8 6.88 387 5CGGN{9}GCC 33 7 6.82 388 6 CCCN{9}CGC 35 8 6.72 389 7 CCCN{1}GCG 34 86.64 390 8 CCCN{0}CAC 103 48 6.61 391 9 AGCN{16}CCG 29 6 5.96 392 10CCCN{4}CGC 34 8 5.8 393 11 CGCN{13}GGA 26 5 5.77 394 12 GCGN{16}CCC 30 75.74 395 13 CGCN{5}GCA 25 5 5.49 396 14 CCCN{14}CCC 101 49 5.43 397 15CTGN{4}CGC 34 9 5.41 398 16 CCAN{12}GCG 28 6 5.37 399 17 CGGN{11}CAG 3610 5.25 400 18 CCCN{5}GCC 75 33 4.87 401 19 GCCN{0}CCC 64 26 4.81 402 20CGCN{4}GAC 19 3 4.78 403 21 CGGN{0}CAG 33 9 4.76 404 22 CCCN{3}CGC 32 84.67 405 23 CGCN{1}GAC 20 3 4.58 406 24 GCGN{2}GCC 29 7 4.54 407 25CCCN{4}GCC 76 34 4.53 408 26 CCCN{1}CCC 103 52 4.53 409 27 CCGN{13}CAG33 9 4.5 410 28 GCCN{4}GGA 64 27 4.48 411 29 CCGN{3}GGA 26 6 4.46 412 30AGGN{2}GGG 118 63 4.44 413 31 CACN{5}GCG 22 4 4.42 414 32 CGCN{17}CCA 276 4.39 415 33 CCCN{9}GGC 69 30 4.38 416 34 CCTN{5}GCG 28 7 4.37 417 35GCGN{0}GAC 19 3 4.32 418 36 GCCN{0}GGC 40 7 4.28 419 37 GCGN{2}CCC 26 64.27 420 38 CCGN{11}CCC 32 9 4.17 421 39 CCCN{8}TCG 23 5 4.12 422 40CCGN{17}GCC 30 8 4.12 423 41 GGGN{5}GGA 101 52 4.11 424 42 GGCN{6}GGA 7132 4.1 425 43 CCAN{4}CCC 96 48 4.1 426 44 CCTN{14}CCG 32 9 4.09 427 45GACN{12}GGC 45 16 4.07 428 46 CGCN{13}CCC 30 8 4.04 429 47 CAGN{16}CCC92 46 4.02 430 48 AGCN{10}GGG 75 35 3.94 431 49 CGGN{13}GGC 30 8 3.93432 50 CGGN{1}GCC 30 8 3.92 433 51 AGCN{0}GGC 26 6 3.9 434 52CCCN{16}GGC 64 28 3.89 435 53 GCTN{19}CCC 67 29 3.87 436 54 CCCN{16}GGG88 31 3.81 437 55 CCCN{9}CGG 30 8 3.77 438 56 CCCN{10}CGG 30 8 3.76 43957 CCAN{0}GCG 32 9 3.75 440 58 GCCN{17}CGC 26 6 3.74 441 59 CCTN{6}CGC27 7 3.73 442 60 GGAN{1}CCC 63 27 3.71 443 61 CGCN{18}CAC 24 5 3.7 44462 CGCN{20}CCG 21 4 3.69 445 63 CCGN{0}GCA 26 6 3.69 446 64 CGCN{20}CCC28 7 3.69 447 65 AGCN{15}CCC 67 30 3.65 448 66 CCTN{7}GGC 69 31 3.63 44967 GCCN{5}CGC 32 9 3.61 450 68 GCCN{14}CGC 28 7 3.59 451 69 CAGN{11}CCC89 45 3.58 452 70 GGGN{16}GAC 53 21 3.57 453 71 CCCN{15}GCG 25 6 3.57454 72 CCCN{0}CGC 37 12 3.54 455 73 CCCN{16}AGC * 67 30 3.54 456 74AGGN{9}GGG 96 50 3.52 457 75 CGCN{12}CTC 28 7 3.46 458 76 CACN{8}CGC 235 3.43 459 77 CCAN{7}CCG 31 9 3.42 460 78 CGGN{1}GCA 25 6 3.41 461 79CGCN{14}CCC 29 8 3.4 462 80 AGCN{0}CCC 76 36 3.4 463 81 CGCN{13}GTC 18 33.37 464 82 GCGN{3}GCA 26 7 3.35 465 83 CGGN{0}GGC 34 11 3.35 466 84GCCN{14}CCC 68 31 3.33 467 85 ACCN{7}CGC 21 4 3.32 468 86 AGGN{7}CGG 3310 3.31 469 87 CCCN{16}CGA 22 5 3.3 470 88 CGCN{6}CAG 31 9 3.29 471 89CAGN{11}GCG 29 8 3.29 472 90 CCGN{12}CCG 19 4 3.26 473 91 CGCN{18}CAG 277 3.24 474 92 CAGN{1}GGG 80 39 3.21 475 93 CGCN{0}CCC 32 10 3.2 476 94GCGN{18}GCC 26 7 3.18 477 95 CGGN{15}GGC 27 7 3.15 478 96 CCCN{15}AGG 7234 3.14 479 97 AGGN{20}GCG 26 7 3.14 480 98 CGGN{5}CTC 26 7 3.13 481 99TCCN{17}CGA 23 5 3.12 482 100 GCGN{4}CCC 30 9 3.08 483 101 CCCN{2}CGC 309 3.07 484 102 CGTN{3}CAG 28 8 3.06 485 103 CCGN{13}GAG 27 7 3.05 486104 CTCN{6}CGC 28 8 3.04 487 105 CGCN{4}GAG 21 5 3.03 488 106 GCGN{5}GGA24 6 3.03 489 107 CCGN{1}CAG 27 7 3.01 490 108 CGCN{11}CCG 18 3 2.99 491109 GCGN{19}CCC 26 7 2.98 492 110 CGCN{18}GAA 21 5 2.98 493 111GGGN{19}GGA 78 39 2.95 494 112 CCAN{1}CGG 24 6 2.94 495 113 CCCN{7}GCG25 6 2.94 496 114 AGGN{10}CCC 84 43 2.92 497 115 CCAN{0}GGG 97 52 2.88498 116 CAGN{10}CCC 82 41 2.87 499 117 CCGN{18}CCG 19 4 2.86 500 118CCGN{18}GGC 26 7 2.85 501 119 CCCN{2}GCG 24 6 2.84 502 120 CGCN{1}GGC 257 2.83 503 121 CCGN{5}GAC 19 4 2.81 504 122 GGAN{0}CCC 52 22 2.8 505 123CCCN{1}CCG 29 9 2.78 506 124 CCCN{15}ACG 23 6 2.75 507 125 AGCN{8}CCC 6631 2.73 508 126 CCCN{3}GGC 60 27 2.71 509 127 AGGN{9}CGG 31 10 2.7 510128 CCCN{14}CGC 27 8 2.7 511 129 CCGN{0}CCG 19 4 2.7 512 130 CGCN{8}AGC23 6 2.69 513 131 CGCN{19}ACC 21 5 2.68 514 132 GCGN{17}GAC 17 3 2.66515 133 AGCN{1}GCG 24 6 2.63 516 134 CCGN{11}GGC 31 10 2.63 517 135CGGN{4}AGA 26 7 2.63 518 136 CGCN{14}CCG 17 3 2.62 519 137 CCTN{20}GCG24 6 2.62 520 138 CCAN{10}CGC 26 7 2.61 521 139 CCCN{20}CAC 69 33 2.6522 140 CCGN{11}GCC 27 8 2.6 523 141 CGCN{18}CCC 26 7 2.59 524 142CGGN{15}CGC 16 3 2.57 525 143 CGCN{16}GCC 24 6 2.55 526 144 CGCN{20}GGC23 6 2.54 527 145 CGCN{19}CCG 18 4 2.52 528 146 CGGN{10}CCA 28 8 2.51529 147 CGCN{17}CCC 26 7 2.51 530 148 CGCN{11}ACA 23 6 2.51 531 149CGGN{0}ACC 17 3 2.5 532 150 GCGN{10}GCC 24 6 2.49 533 151 GCGN{8}GAC 173 2.49 534 152 CCCN{15}GGG 84 32 2.44 535 153 CGGN{16}GGC 27 8 2.44 536154 CGCN{16}CCA 23 6 2.42 537 155 GCCN{3}CCC 73 36 2.4 538 156CAGN{4}GGG 94 51 2.4 539 157 CCCN{6}GCG 23 6 2.38 540 158 CCGN{16}CGC 173 2.38 541 159 CCCN{17}GCA 61 28 2.37 542 160 CGCN{13}TCC 24 6 2.37 543161 GCCN{1}CGC 29 9 2.36 544 162 CCGN{19}GAG 26 7 2.35 545 163GGGN{10}GGA 89 48 2.35 546 164 CAGN{5}CCG 32 11 2.35 547 165 CGCN{3}AGA19 4 2.32 548 166 GCCN{0}GCC 29 9 2.32 549 167 CCCN{8}GGC 61 28 2.31 550168 CCTN{6}GCG 22 6 2.29 551 169 GACN{6}CCC 48 20 2.29 552 170CGGN{1}CCC 26 8 2.27 553 171 CCCN{15}CCG 30 10 2.27 554 172 CAGN{9}CCC84 44 2.26 555 173 CGGN{10}GGC 27 8 2.26 556 174 CGAN{10}ACG 10 1 2.26557 175 GCGN{3}TCC 21 5 2.26 558 176 CCCN{3}GCC 75 38 2.24 559 177GCGN{1}ACC 17 3 2.24 560 178 CCGN{9}AGG 27 8 2.23 561 179 CGCN{16}CAG 268 2.23 562 180 GGCN{0}CCC 62 29 2.22 563 181 AGGN{12}CCG 26 8 2.19 564182 CCGN{0}GCG 16 3 2.19 565 183 CCGN{2}GCC 30 10 2.18 566 184CCGN{11}GTC 19 4 2.17 567 185 CAGN{0}CCC 88 47 2.17 568 186 CCCN{5}CCG32 11 2.17 569 187 GCCN{20}CCC 66 32 2.15 570 188 GACN{2}CGC 18 4 2.14571 189 CGCN{6}CAC 23 6 2.13 572 190 AGGN{14}GCG 25 7 2.1 573 191GACN{5}CGC 17 3 2.1 574 192 CCTN{19}CCG 29 9 2.1 575 193 CCGN{12}GGA 247 2.08 576 194 GGCN{9}GAC * 44 18 2.08 577 195 AGGN{10}GGG 94 52 2.07578 196 CCGN{10}GAG 25 7 2.07 579 197 CGCN{6}GGA 20 5 2.06 580 198CGCN{7}AGC 23 6 2.04 581 199 CCAN{13}CGG 26 8 2.03 582 200 CGGN{6}GGA 257 2.03 583 201 CGCN{19}GCC 24 7 2.03 584 202 CCAN{12}CGC 24 7 2.02 585203 CGGN{1}GGC 41 16 2.02 586 204 GCGN{3}CCA 25 7 2.01 587 205AGGN{1}CGC 21 5 2 588 206 CTCN{5}CGC 24 7 1.98 589 207 CCCN{0}ACG 30 101.97 590 208 CAGN{17}CCG 29 9 1.96 591 209 GGCN{4}CCC 62 30 1.96 592 210AGGN{8}GCG 26 8 1.96 593 211 CTGN{1}CCC 88 48 1.94 594 212 CCCN{16}CAG85 46 1.94 595 213 CGCN{9}GAC 16 3 1.93 596 214 CAGN{6}CCG 29 9 1.92 597215 CGTN{12}CGC 11 1 1.92 598 216 CTCN{7}GCC 69 35 1.92 599 217CGCN{19}TCC 22 6 1.92 600 218 CCCN{7}GCC 67 33 1.91 601 219 CAGN{13}CGG30 10 1.9 602 220 CGCN{1}GCC 27 8 1.9 603 221 CGCN{17}CCG 17 4 1.89 604222 AGGN{4}CCC 63 31 1.89 605 223 AGCN{10}CGC 21 5 1.89 606 224CCCN{11}CGG 30 10 1.88 607 225 CCCN{8}GCC 75 39 1.86 608 226 CCGN{1}CGG22 3 1.86 609 227 CCCN{1}ACC 71 36 1.85 610 228 CGCN{0}CAG 25 7 1.85 611229 CCGN{19}TGC 23 6 1.82 612 230 GCGN{4}CGA 12 2 1.82 613 231CCGN{19}GCC 30 10 1.82 614 232 CCAN{10}CCC 85 46 1.81 615 233CAGN{13}GGG 91 51 1.81 616 234 AGCN{18}CGG 23 6 1.81 617 235 CGAN{8}CGC11 1 1.81 618 236 AGCN{4}CCC 63 31 1.8 619 237 GGAN{6}CCC 61 30 1.8 620238 CGGN{13}AAG 23 6 1.8 621 239 ACCN{11}CGC 19 5 1.79 622 240CCGN{12}CAG 28 9 1.78 623 241 CCCN{12}GGG 76 29 1.77 624 242 CACN{17}ACG22 6 1.76 625 243 CAGN{18}CCC 82 44 1.76 626 244 CGTN{10}GTC 19 5 1.75627 245 CCCN{13}GCG 23 6 1.75 628 246 GCAN{1}CGC 20 5 1.73 629 247AGAN{4}CCG 24 7 1.73 630 248 GCGN{10}AGC 22 6 1.72 631 249 CGCN{0}GGA 122 1.72 632 250 CGGN{4}GAC 17 4 1.69 633 251 CCCN{12}CGC 26 8 1.68 634252 GCCN{15}CCC 65 33 1.68 635 253 GCGN{6}TCC 20 5 1.66 636 254CGGN{3}CAG 33 12 1.65 637 255 CCCN{3}CCA 88 49 1.65 638 256 AGCN{3}CCC59 28 1.65 639 257 GGGN{16}GCA 65 33 1.65 640 258 AGGN{8}CCG 28 9 1.64641 259 CCCN{0}CCG 29 10 1.64 642 260 GCGN{5}GAC 16 3 1.64 643 261CCCN{9}ACC 60 29 1.64 644 262 CTGN{5}CGC 25 8 1.64 645 263 CGCN{14}CTC23 7 1.64 646 264 CGGN{14}GCA 23 7 1.63 647 265 CCGN{8}GCC 26 8 1.62 648266 CCGN{7}CAC 23 7 1.62 649 267 AGCN{8}GCG 21 6 1.61 650 268CGGN{16}GGA 29 10 1.61 651 269 CCAN{12}CCG 26 8 1.61 652 270 CGGN{2}CCC26 8 1.6 653 271 CCAN{13}GGG 71 37 1.6 654 272 CGGN{15}GCA 21 6 1.6 655273 CGCN{9}GCA 20 5 1.58 656 274 CGGN{19}CCA 26 8 1.58 657 275GGGN{15}CGA 20 5 1.57 658 276 CCCN{10}CGC 26 8 1.57 659 277 CTCN{14}CGC26 8 1.55 660 278 CACN{11}GCG 20 5 1.55 661 279 CCGN{2}GGC 24 7 1.55 662280 CTGN{18}CCC 85 47 1.54 663 281 GGGN{13}CAC 58 28 1.54 664 282CCTN{15}GGC 62 31 1.54 665 283 CCCN{20}CGA 20 5 1.54 666 284 CCCN{8}CGA20 5 1.53 667 285 GAGN{7}CCC 61 30 1.53 668 286 CGCN{2}CCG 22 6 1.53 669287 CCCN{0}TCC 98 57 1.52 670 288 AGCN{0}GCC 21 6 1.52 671 289CCCN{2}TCC 82 45 1.52 672 290 CCGN{5}CCC 30 10 1.52 673 291 CGCN{13}CGC16 3 1.51 674 292 CCCN{1}CGC 28 9 1.51 675 293 GCCN{16}GCA 53 25 1.51676 294 CCCN{16}CCA 84 46 1.5 677 295 CCGN{13}CGC 19 5 1.5 678 296CCGN{17}CAG 28 9 1.49 679 297 CGGN{18}GGC 26 8 1.49 680 298 CCGN{14}AGG23 7 1.49 681 299 CCCN{5}CGG 26 8 1.49 682 300 CCCN{6}GGA 58 28 1.49 683301 ACGN{2}CCC 20 5 1.49 684 302 CCAN{9}CCG 27 9 1.48 685 303CCCN{19}CCA 78 42 1.48 686 304 CAGN{0}GGG 77 41 1.48 687 305 AGCN{1}CCC58 28 1.47 688 306 GCGN{7}TCC 27 9 1.46 689 307 ACGN{18}CCA 25 8 1.46690 308 GCTN{14}CCC 61 30 1.46 691 309 GCGN{14}CCC 23 7 1.46 692 310GCGN{19}AGC 20 5 1.45 693 311 CCGN{8}CAG 29 10 1.45 694 312 GCGN{6}GCC22 6 1.45 695 313 GCGN{10}GCA 20 5 1.44 696 314 CCTN{7}GCC 69 36 1.44697 315 GCCN{13}GCC 54 26 1.42 698 316 CCCN{14}GCC 63 32 1.42 699 317CCCN{15}CGG 26 8 1.42 700 318 CCAN{13}CGC 23 7 1.42 701 319 AGCN{11}GGG67 35 1.41 702 320 GGAN{0}GCC 64 32 1.4 703 321 GCCN{3}TCC 61 30 1.4 704322 CCTN{5}GCC 69 36 1.39 705 323 CGGN{18}CCC 25 8 1.39 706 324CCTN{3}GGC 59 29 1.38 707 325 CCGN{0}CTC 22 6 1.38 708 326 AGCN{17}GCG19 5 1.37 709 327 ACGN{14}GGG 20 5 1.37 710 328 CGAN{12}GGC 19 5 1.37711 329 CCCN{20}CGC 24 7 1.37 712 330 ACGN{12}CTG 24 7 1.36 713 331CCGN{0}CCC 36 14 1.36 714 332 CCGN{10}GGA 23 7 1.36 715 333 CCCN{3}GCG21 6 1.36 716 334 GCGN{14}CGC 22 3 1.35 717 335 CCGN{8}CGC 16 4 1.35 718336 CGCN{10}ACA 22 6 1.34 719 337 CCCN{19}CCG 28 10 1.33 720 338CACN{14}CGC 20 5 1.32 721 339 GACN{3}GGC 46 21 1.32 722 340 GAAN{7}CGC19 5 1.32 723 341 CGCN{16}GGC 21 6 1.31 724 342 GGCN{9}CCC 64 33 1.31725 343 CCCN{9}GCC 64 33 1.31 726 344 CGCN{0}TGC 26 9 1.3 727 345CCTN{8}GGC 67 35 1.3 728 346 CCAN{8}CCC 82 46 1.29 729 347 GACN{2}CCC 4218 1.28 730 348 GGCN{1}CCC 54 26 1.27 731 349 CGCN{0}AGC 24 7 1.26 732350 AGGN{4}GCG 28 10 1.26 733 351 CGGN{6}TCC 22 6 1.25 734 352ACGN{19}GGC 20 5 1.25 735 353 CCCN{8}ACG 21 6 1.24 736 354 CCCN{18}GCC62 31 1.24 737 355 GCCN{2}CGA 19 5 1.24 738 356 CCCN{8}GCG 28 10 1.23739 357 CCCN{0}CTC 76 41 1.23 740 358 GCCN{11}CGC 27 9 1.22 741 359AGCN{9}CCC 59 29 1.22 742 360 GCTN{0}GCC 71 38 1.21 743 361 CGCN{3}CCC26 9 1.21 744 362 CCCN{2}CCC 117 72 1.19 745 363 GCCN{9}CGC 23 7 1.19746 364 GCAN{19}CGC 19 5 1.19 747 365 CAGN{4}CGG 32 12 1.18 748 366CAGN{2}GGG 80 44 1.17 749 367 GCCN{16}CCC 67 35 1.16 750 368 GAGN{5}CCC60 30 1.16 751 369 CCTN{16}TCG 20 6 1.16 752 370 CCCN{2}GGC 62 32 1.15753 371 GCGN{13}GGA 24 8 1.15 754 372 GCCN{17}GGC 66 25 1.15 755 373CCCN{14}GGC 58 29 1.14 756 374 AGGN{3}CCG 31 12 1.14 757 375 CACN{0}CGC32 12 1.14 758 376 CGGN{18}CAG 28 10 1.14 759 377 AGCN{1}GCC 57 28 1.13760 378 CGCN{18}GGC 23 7 1.13 761 379 CCCN{5}AGG 64 33 1.11 762 380AACN{0}GCG 9 1 1.11 763 381 CCCN{10}CCA 88 50 1.09 764 382 CGCN{13}GAG20 6 1.09 765 383 CGCN{7}GCC 25 8 1.08 766 384 CCCN{9}CCG 28 10 1.07 767385 CGCN{16}CCC 24 8 1.05 768 386 GAAN{13}CGC 18 5 1.05 769 387GGCN{3}CCC 49 23 1.03 770 388 TCCN{11}CCA 87 50 1.03 771 389 CACN{0}CCC70 38 1.02 772 390 CGCN{16}CCG 15 3 1.02 773 391 CGGN{15}AGC 21 6 1.02774 392 CCCN{12}GCG 21 6 1.02 775 393 CCCN{9}GAG 59 30 1.01 776 394CCGN{20}TCC 24 8 1.01 777 395 CGCN{0}CGC 17 4 1.01 778 396 ATGN{7}CGG 206 1 779 397 GGGN{20}GCA 59 30 1 780 398 CGGN{4}GGC 26 9 0.99 781 399CGGN{16}AGC 22 7 0.99 782 400 CGGN{5}GGC 25 8 0.99 783 401 GCGN{0}GGA 258 0.98 784 402 GGCN{20}CAC 52 25 0.98 785 403 CCCN{9}CCC 97 58 0.97 786404 ACCN{17}GGC 44 20 0.97 787 405 CCCN{6}CGA 18 5 0.96 788 406AAGN{10}CGG 26 9 0.96 789 407 CGCN{17}CAC 21 6 0.95 790 408 CCCN{16}CGG25 8 0.94 791 409 GACN{18}GGC 39 17 0.94 792 410 GGGN{15}GAC 47 22 0.92793 411 GCCN{4}TCC 66 35 0.92 794 412 GGCN{15}CCC 56 28 0.92 795 413CAGN{12}CGC 24 8 0.92 796 414 CCAN{3}GCG 22 7 0.91 797 415 CCGN{16}GAG22 7 0.9 798 416 AGCN{2}CGC 24 8 0.89 799 417 GAGN{4}CCC 54 27 0.89 800418 AGGN{3}CGC 23 7 0.88 801 419 CACN{13}AGG * 67 36 0.88 802 420CCCN{4}CAG 88 51 0.88 803 421 CCCN{2}GAA 63 33 0.87 804 422 CGCN{19}GAG21 6 0.87 805 423 ACGN{18}GGG 21 6 0.87 806 424 CCCN{4}GGC 62 32 0.87807 425 CGGN{9}GAG 28 10 0.86 808 426 CCCN{3}GGG 66 26 0.86 809 427GAGN{4}GGC 66 35 0.85 810 428 CGCN{5}GAG 18 5 0.84 811 429 CCGN{20}AGG24 8 0.84 812 430 CCCN{15}CCC 88 51 0.83 813 431 AGGN{17}CCG 25 8 0.82814 432 AGGN{6}GGG 89 52 0.82 815 433 GGCN{20}CCC 57 29 0.82 816 434GCAN{17}CGC 19 5 0.82 817 435 CGAN{11}ACG 9 1 0.81 818 436 CGCN{2}GGA 195 0.81 819 437 CTGN{5}CCC 79 45 0.8 820 438 TCCN{20}CCA 77 43 0.8 821439 CCAN{2}GGG 59 30 0.8 822 440 CCGN{15}GCG 14 3 0.8 823 441 CCAN{5}GGG69 38 0.79 824 442 CGGN{1}TGC 24 8 0.79 825 443 CCCN{14}GCG 21 6 0.79826 444 CAGN{0}CCG 27 10 0.79 827 445 GCCN{9}TCC 60 31 0.78 828 446AGGN{20}CGC 22 7 0.78 829 447 CCCN{6}GAC 42 19 0.77 830 448 CGGN{11}CCA23 7 0.76 831 449 GGGN{14}CAC 57 29 0.75 832 450 GCAN{15}CGC 19 5 0.74833 451 CGCN{2}ACA 20 6 0.74 834 452 ACCN{9}CCC 57 29 0.73 835 453GCGN{9}CGC 20 3 0.73 836 454 CAGN{15}GCG 23 7 0.73 837 455 CCCN{18}GTC45 21 0.72 838 456 GCGN{3}CCC 24 8 0.72 839 457 CGGN{11}GCC 23 8 0.72840 458 CCCN{1}CGG 24 8 0.71 841 459 GCCN{4}CCA 70 38 0.71 842 460CCCN{4}CCG 30 12 0.7 843 461 CGTN{2}GCA 21 6 0.7 844 462 AGCN{7}TCG 18 50.69 845 463 CCGN{15}GAA 20 6 0.69 846 464 ACCN{5}CCC 62 33 0.69 847 465CGCN{14}GAG 19 5 0.68 848 466 CCCN{7}CGC 30 12 0.68 849 467 GAGN{12}CGC21 6 0.68 850 468 GGCN{17}CCC 58 30 0.67 851 469 ACGN{11}CTC 21 7 0.65852 470 ACAN{9}CGG 24 8 0.65 853 471 CTGN{7}CCC 82 47 0.65 854 472CCCN{2}GCC 72 40 0.65 855 473 CGGN{2}GCA 24 8 0.64 856 474 CCCN{0}TGC 8348 0.64 857 475 CGCN{7}ACC 18 5 0.63 858 476 GCAN{2}GCC 54 27 0.63 859477 GCGN{8}CCA 20 6 0.63 860 478 AGCN{0}CGC 22 7 0.63 861 479 GCGN{2}GCA18 5 0.63 862 480 CCGN{2}GTC 18 5 0.62 863 481 CCGN{3}ACA 21 7 0.62 864482 ACGN{13}TGG 21 7 0.62 865 483 CCAN{8}CGC 23 8 0.62 866 484CCGN{9}GGC 23 8 0.61 867 485 CCAN{5}CCG 25 9 0.61 868 486 AGGN{3}GGG 9759 0.61 869 487 CAGN{2}GGC 78 45 0.61 870 488 CCCN{8}CAG 81 47 0.61 871489 AGCN{5}CAG 80 46 0.6 872 490 CGGN{16}GCC 22 7 0.6 873 491GCGN{15}CCC 23 8 0.6 874 492 CCCN{11}GCC 59 31 0.59 875 493 CGAN{2}ACG 91 0.59 876 494 CGGN{4}GCC 22 7 0.59 877 495 CACN{6}CGC 19 6 0.59 878 496CGGN{5}ACG 11 2 0.59 879 497 CTGN{4}GCC * 66 36 0.59 880 498 GGGN{18}CGA18 5 0.59 881 499 CCTN{8}CGC 22 7 0.59 882 500 GCCN{4}CCC 67 37 0.58 883501 CGGN{10}GCC 22 7 0.58 884 502 GCCN{5}GGA 54 27 0.57 885 503ACCN{7}GCG 15 4 0.57 886 504 CCCN{8}CGC 24 8 0.57 887 505 CAGN{5}CCC 7744 0.56 888 506 CACN{14}GGA 63 34 0.56 889 507 CCCN{1}GCC 94 57 0.55 890508 CCCN{5}AGC 67 37 0.55 891 509 GGCN{5}GGA 59 31 0.55 892 510CGAN{17}GAG 19 6 0.55 893 511 CGCN{7}ACA 18 5 0.54 894 512 CCAN{13}CCC87 52 0.54 895 513 CGGN{20}GGC 24 8 0.54 896 514 CCCN{17}GCC 58 30 0.53897 515 CCTN{10}CCG 30 12 0.53 898 516 CCCN{8}CCG 27 10 0.53 899 517CGCN{3}GAG 18 5 0.52 900 518 CGCN{7}AAG 17 5 0.51 901 519 CGGN{11}GGA 238 0.51 902 520 CCGN{15}CCG 15 4 0.51 903 521 CCCN{3}GCA 57 30 0.51 904522 CGGN{2}CAG 24 8 0.5 905 523 AGGN{2}CCG 24 8 0.5 906 524 CCCN{4}CAC69 38 0.5 907 525 GGAN{19}CCC 56 29 0.49 908 526 CCCN{8}CAC 68 38 0.49909 527 ACCN{6}CCG 18 5 0.49 910 528 CCCN{6}GGC 54 28 0.49 911 529CCCN{6}CCG 29 11 0.48 912 530 CGCN{14}GCC 26 9 0.47 913 531 CCGN{5}TCC25 9 0.46 914 532 GCCN{6}GCC 55 28 0.46 915 533 CGGN{7}GGA 24 8 0.45 916534 GGGN{6}GGA 87 52 0.44 917 535 GCCN{12}TCC 60 32 0.44 918 536AGTN{16}CCG 17 5 0.44 919 537 GGCN{19}GCC 68 29 0.44 920 538 CCGN{3}CCG22 7 0.44 921 539 CCCN{8}ACC 58 31 0.44 922 540 CAGN{15}GCC 77 44 0.44923 541 CCCN{17}CGG 24 8 0.44 924 542 GCGN{1}CCA 22 7 0.44 925 543CCCN{14}CAG 79 46 0.44 926 544 CCCN{8}CCC 89 53 0.44 927 545 ACAN{12}GCG23 8 0.43 928 546 AGGN{4}CCG 23 8 0.43 929 547 CGCN{13}GCC 23 8 0.43 930548 GAGN{2}CGC 23 8 0.42 931 549 CCCN{9}GCG 21 7 0.42 932 550CGCN{17}ACA 17 5 0.42 933 551 GCGN{17}CCA 23 8 0.42 934 552 AAGN{18}CCG20 6 0.42 935 553 CGCN{1}GGA 18 5 0.41 936 554 CCAN{1}CCC 90 54 0.41 937555 CGTN{18}TGC 20 6 0.41 938 556 TCCN{14}CGA 17 5 0.41 939 557CACN{5}GGG 56 29 0.4 940 558 CCGN{12}GCA 21 7 0.4 941 559 CTGN{6}CCC 7744 0.4 942 560 CGGN{8}GGC 32 13 0.4 943 561 CCAN{11}GGG 68 38 0.4 944562 ACGN{19}CAA 21 7 0.39 945 563 GGGN{20}CCC 72 31 0.39 946 564CGCN{3}CAG 23 8 0.39 947 565 AGCN{17}GGG 58 31 0.37 948 566 CACN{20}CCG21 7 0.37 949 567 ACGN{17}CAG 24 8 0.37 950 568 AGGN{1}CCC 60 32 0.37951 569 CGTN{12}CAC 20 6 0.37 952 570 CGGN{9}GGC 23 8 0.37 953 571CGCN{10}GCG 18 3 0.37 954 572 CCCN{6}CTC 80 47 0.36 955 573 CCGN{10}AGG23 8 0.36 956 574 CCCN{18}CAG 79 46 0.36 957 575 AGCN{17}CCG 21 7 0.36958 576 AGCN{9}GCG 18 5 0.36 959 577 CCAN{3}GGC 62 34 0.36 960 578CCCN{11}GGC 57 30 0.35 961 579 ACGN{5}GCA 23 8 0.35 962 580 CCCN{14}CGG23 8 0.35 963 581 CCCN{5}CCA 91 55 0.35 964 582 CCGN{1}AGG 22 7 0.34 965583 GGGN{10}GAC 45 22 0.34 966 584 CGCN{15}CCA 20 6 0.34 967 585CCTN{19}CGC 22 7 0.34 968 586 CGTN{3}CGC 10 2 0.33 969 587 AGCN{14}CCG21 7 0.33 970 588 GGCN{2}CGA 17 5 0.33 971 589 CAGN{8}CCC 79 46 0.33 972590 CCGN{2}GAC 16 4 0.33 973 591 AGCN{19}AGG 70 40 0.32 974 592CCTN{4}GGC 64 35 0.32 975 593 CCGN{11}AGC 22 7 0.32 976 594 CACN{4}CGC18 5 0.32 977 595 CCGN{1}CCC 30 12 0.31 978 596 CTGN{13}GGC 73 42 0.31979 597 CGCN{16}ACC 15 4 0.31 980 598 CACN{18}CAG 79 46 0.31 981 599GGCN{8}GCC 68 29 0.29 982 600 GGGN{15}GGA 78 46 0.29 983 601 CCGN{16}GCC22 7 0.29 984 602 CCGN{20}ACC 18 5 0.29 985 603 CGAN{7}CCC 17 5 0.28 986604 CCGN{6}CTC 23 8 0.28 987 605 CGGN{10}CTC 22 7 0.28 988 606CAGN{16}CGC 23 8 0.28 989 607 CCAN{3}AGG 77 45 0.27 990 608 GCCN{18}GCC52 27 0.27 991 609 CGCN{18}GGA 19 6 0.26 992 610 CCGN{20}GGC 22 7 0.26993 611 ACAN{10}GCG 17 5 0.26 994 612 CGGN{5}CCC 25 9 0.25 995 613CCCN{7}TCC 75 43 0.25 996 614 ACGN{10}CGC 10 2 0.25 997 615 CCCN{3}TCC81 48 0.25 998 616 CCGN{8}CGG 20 3 0.24 999 617 CCAN{15}CGG 22 7 0.241000 618 CCGN{6}CCG 17 5 0.24 1001 619 CAGN{3}GCG 25 9 0.24 1002 620GAGN{1}CCC 62 34 0.24 1003 621 CCGN{18}TGC 22 7 0.23 1004 622 CCCN{7}CCA85 51 0.23 1005 623 CGGN{3}CCA 24 9 0.23 1006 624 ACGN{1}CCC 18 5 0.231007 625 CGGN{13}TGA 21 7 0.22 1008 626 CTCN{6}GGC 53 28 0.22 1009 627GCGN{2}GAC 15 4 0.22 1010 628 GGGN{11}ACC 49 25 0.22 1011 629 CGCN{4}GGA17 5 0.22 1012 630 CCCN{11}CCG 27 10 0.22 1013 631 CCGN{19}GCA 20 6 0.221014 632 GCGN{0}GCA 20 6 0.21 1015 633 AGAN{7}CCC 61 33 0.21 1016 634CGGN{2}CCA 21 7 0.21 1017 635 CCCN{7}CCC 89 54 0.21 1018 636 ACCN{4}GCG15 4 0.2 1019 637 CCTN{15}CGC 20 6 0.2 1020 638 AGCN{9}GTC 44 21 0.21021 639 CCCN{18}CTC 74 43 0.2 1022 640 CGCN{18}CGA 9 1 0.19 1023 641CCCN{15}GCC 62 34 0.18 1024 642 ACCN{11}GGC 45 22 0.18 1025 643AGGN{15}CGC 29 12 0.18 1026 644 GCGN{0}CCA 27 10 0.18 1027 645GCGN{9}AGC 18 5 0.17 1028 646 GGGN{18}GCA 59 32 0.17 1029 647CCCN{17}CAG 77 45 0.17 1030 648 CCAN{8}CGG 22 8 0.16 1031 649CCGN{10}GGC 21 7 0.16 1032 650 GCAN{0}GCC 76 44 0.16 1033 651 CAGN{2}CGC20 6 0.16 1034 652 CGCN{8}GGC 19 6 0.16 1035 653 CTGN{17}GGC 65 36 0.161036 654 GGGN{14}ACC 46 23 0.16 1037 655 CCGN{1}TGC 20 6 0.16 1038 656CAGN{8}CGC 22 8 0.15 1039 657 AAGN{11}CGC 17 5 0.15 1040 658 CCGN{6}TCC22 8 0.14 1041 659 CCAN{18}CCC 72 42 0.14 1042 660 CCAN{0}CCC 84 51 0.141043 661 GAGN{6}CCC 53 28 0.14 1044 662 AGCN{20}GGC 52 27 0.14 1045 663CAGN{0}CGC 21 7 0.14 1046 664 CCGN{12}CTC 22 8 0.14 1047 665 CGCN{15}ACG9 1 0.13 1048 666 GGCN{17}CGA 15 4 0.13 1049 667 CCGN{16}AAG 19 6 0.131050 668 CGCN{14}TCC 19 6 0.12 1051 669 AGGN{7}CGC 20 7 0.12 1052 670CGGN{7}CCC 22 8 0.12 1053 671 CGCN{4}GCC 34 15 0.12 1054 672 CGAN{6}CCC17 5 0.12 1055 673 CCCN{19}GGA 60 33 0.11 1056 674 CCCN{16}GCG 28 110.11 1057 675 CCAN{7}CGC 20 7 0.11 1058 676 CCCN{6}GCC 80 48 0.11 1059677 GCCN{14}TCC 55 29 0.11 1060 678 AGGN{14}GCC 64 36 0.1 1061 679CGCN{11}GCC 20 7 0.1 1062 680 TCCN{0}GCA 17 5 0.09 1063 681 GCGN{8}CCC27 11 0.09 1064 682 CCAN{11}GCG 19 6 0.09 1065 683 CACN{4}GGG 51 26 0.091066 684 CGGN{7}TCC 20 7 0.09 1067 685 GCGN{5}GCC 20 7 0.09 1068 686ACGN{12}CAG 26 10 0.09 1069 687 CCGN{19}CGC 14 4 0.08 1070 688CGGN{8}TGC 18 5 0.08 1071 689 CCCN{1}GAG 65 37 0.07 1072 690 GCGN{19}TGA18 6 0.07 1073 691 GGCN{15}GCC 70 31 0.07 1074 692 CCGN{7}CCC 27 11 0.071075 693 ACAN{19}CCC 63 35 0.07 1076 694 ACCN{16}GGG 47 24 0.07 1077 695AGAN{1}GGC 64 36 0.07 1078 696 GGGN{17}TGA 64 36 0.06 1079 697CAGN{5}GGG 83 50 0.06 1080 698 GCCN{13}CGC 22 8 0.06 1081 699 GCGN{7}GGA19 6 0.06 1082 700 CAGN{14}CCA 94 58 0.06 1083 701 CCGN{4}GTC 16 4 0.061084 702 CCCN{13}CGC 22 8 0.06 1085 703 GCGN{14}ACC 15 4 0.05 1086 704CAGN{20}GGG 81 49 0.05 1087 705 CCGN{4}CCC 27 11 0.05 1088 706CGCN{5}GGC 18 6 0.05 1089 707 CCTN{6}GGC 57 31 0.05 1090 708 AGGN{3}GGC67 38 0.05 1091 709 CGGN{11}CGC 14 4 0.05 1092 710 CTGN{18}GGA 77 460.04 1093 711 CACN{17}CCA 74 43 0.04 1094 712 CGGN{3}GAG 22 8 0.04 1095713 CCCN{9}CCA 82 49 0.03 1096 714 CCCN{1}ACG 18 6 0.03 1097 715CAGN{1}GCC 72 42 0.03 1098 716 AGGN{6}CCG 23 8 0.03 1099 717 AGCN{9}GGG57 31 0.03 1100 718 CCCN{7}GGC 54 29 0.02 1101 719 CCTN{13}CCC 88 540.02 1102 720 CCGN{19}TTC 20 7 0.02 1103 721 CCCN{7}CCG 27 11 0.02 1104722 CGAN{6}GGC 17 5 0.01 1105 723 CGGN{4}CTC 21 7 0.01 1106 724CGGN{0}CGC 13 3 0.01 1107 725 CCTN{13}ACG 19 6 0.01 1108 726 GGGN{6}CAC53 28 0.01 1109 727 CCCN{16}CGC 21 7 0.01 1110 728 CCCN{10}CTC 76 45 01111 729 CCCN{0}CAG 92 57 0 1112 730 GCCN{5}CCC 65 37 0 1113

TABLE 6 STAR elements, including genomic location and length (SEQ IDNOS: 1-66) STAR Location¹ Length² SEQ ID NO:  1 2q31.1 750 1  2 7p15.2916 2  3³ 15q11.2 and 10q22.2 2132 3  4 1p31.1 and 14q24.1 1625 4  5⁴20q13.32 1571 5  6 2p21 1173 6  7 1q34 2101 7  8 9q32 1839 8  9⁴ 10p15.31936 9 10 Xp11.3 1167 10 11 2p25.1 1377 11 12 5q35.3 1051 12 13⁴ 9q34.31291 13 14⁴ 22q11.22 732 14 15 1p36.31 1881 15 16 1p21.2 1282 16 172q31.1 793 17 18 2q31.3 497 18 19 6p22.1 1840 19 20 8p13.3 780 20 216q24.2 620 21 22 2q12.2 1380 22 23 6p22.1 1246 23 24 1q21.2 948 24 25⁵1q21.3 1067 25 26 1q21.1 540 26 27 1q23.1 1520 27 28 22q11.23 961 28 292q13.31 2253 29 30 22q12.3 1851 30 31 9q34.11 and 22q11.21 1165 31 3221q22.2 771 32 33 21q22.2 1368 33 34 9q34.14 755 34 35 7q22.3 1211 35 3621q22.2 1712 36 37 22q11.23 1331 37 38 22q11.1 and 22q11.1 ~1000 38 3922q12.3 2331 39 40 22q11.21 1071 40 41 22q11.21 1144 41 42 22q11.1 73542 43 14q24.3 1231 43 44 22q11.1 1591 44 45 22q11.21 1991 45 46 22q11.231871 46 47 22q11.21 1082 47 48 22q11.22 1242 48 49 Chr 12 random clone,and 3q26.32 1015 49 50 6p21.31 2361 50 51 5q21.3 2289 51 52 7p15.2 120052 53 Xp11.3 1431 53 54 4q21.1 981 54 55 15q13.1 501 55 56 includes3p25.3 741 56 57 4q35.2 1371 57 58 21q11.2 1401 58 59 17 random clone872 59 60 4p16.1 and 6q27 2068 60 61 7p14.3 and 11q25 1482 61 62 14q24.31011 62 63 22q13.3 1421 63 64 17q11.2 1414 64 65 7q21.11 = 28.4 1310 6566 20q13.33 and 6q14.1 ~2800 66 ¹Chromosomal location is determined byBLAST search of DNA sequence data from the STAR elements against thehuman genome database. The location is given according to standardnomenclature referring to the cytogenetic ideogram of each chromosome;e.g., 1p2.3 is the third cytogenetic sub-band of the second cytogeneticband of the short arm of chromosome 1(WorldWideWeb.ncbi.nlm.nih.gov/Class/MLACourse/Genetics/chrombanding.html).In cases where the forward and reverse sequencing reaction identifiedDNAs from different genomic loci, both loci are shown. ²Precise lengthsare determined by DNA sequence analysis; approximate lengths aredetermined by restriction mapping. ³Sequence and location of STAR3 (SEQID NO: 3) has been refined since assembly of Tables 2 and 4 of EP01202581.3. ⁴The STARs with these numbers in Tables 2 and 4 of EP01202581.3 have been set aside (hereafter referred to as “oldSTAR5”etc.) and their numbers assigned to the STAR elements shown in the DNAsequence appendix. In the case of oldSTAR5, oldSTAR14, and oldSTAR16,the cloned DNAs were chimeras from more than two chromosomal locations;in the case of oldSTAR9 and oldSTAR13, the cloned DNAs were identical toSTAR4 (SEQ ID NO: 4). ⁵Identical to Table 4 “STAR18” (SEQ ID NO: 18) ofEP 01202581.3.

TABLE 7 STAR elements convey stability over time on transgeneexpression¹ Cell Divisions² Luciferase Expression³ STAR6 (SEQ ID NO: 6)42 18,000 plus puromycin 60 23,000 84 20,000 108 16,000 STAR6 (SEQ IDNO: 6) 84 12,000 without puromycin⁴ 108 15,000 144 12,000 ¹PlasmidpSDH-Tet-STAR6 was transfected into U-2 OS cells, and clones wereisolated and cultivated in doxycycline-free medium. Cells weretransferred to fresh culture vessels weekly at a dilution of 1:20. ²Thenumber of cell divisions is based on the estimation that in one week theculture reaches cell confluence, which represents ~6 cell divisions.³Luciferase was assayed as described in Example 4. ⁴After 60 celldivisions the cells were transferred to two culture vessels; one wassupplied with culture medium that contained puromycin, as for the first60 cell divisions, and the second was supplied with culture mediumlacking antibiotic.

TABLE 8 Human STAR elements and their putative mouse orthologs andparalogs Number STAR Human¹ Mouse² Similarity³ SEQ ID NO: 1  1 2q31.1 2D600 bp 69% 1 2  2 7p15.2 6B3 909 bp 89% 2 3  3a 5q33.3 11B2 248 bp 83% 34  3b 10q22.2 14B 1. 363 bp 89% 3 2. 163 bp 86% 5  6 2p21 17E4 437 bp78% 6 6 12 5q35.3 11b1.3 796 bp 66% 12 7 13 9q34.3 2A3 753 bp 77% 13 818 2q31.3 2E1 497 bp 72% 18 9 36 21q22.2 16C4 166 bp 79% 36 10 4022q11.1 6F1 1. 270 bp 75% 40 2. 309 bp 70% 11 50 6p21.31 17B1 1. 451 bp72% 50 2. 188 bp 80% 3. 142 bp 64% 12 52 7p15.2 6B3 1. 846 bp 74% 52 2.195 bp 71% 13 53 Xp11.3 XA2 364 bp 64% 53 14 54 4q21.1 5E3 1. 174 bp 80%54 2. 240 bp 73% 3. 141 bp 67% 4. 144 bp 68% 15  61a 7p14.3 6B3 188 bp68% 61 ¹Cytogenetic location of STAR element in the human genome.²Cytogenetic location of STAR element ortholog in the mouse genome.³Length of region(s) displaying high sequence similarity, and percentagesimilarity. In some cases more than one block of high similarity occurs;in those cases, each block is described separately. Similarity <60% isnot considered significant.

TABLE 9 Candidate STAR elements tested by Linear Discriminant Analysis(SEQ ID NOS: 66-84) Candidate STAR Location¹ Length SEQ ID NO: T2 F20q13.33 ~2800 66 T2 R 6q14.1 ~2800 67 T3 F 15q12 ~2900 68 T3 R 7q31.2~2900 69 T5 F 9q34.13  ND² 70 T5 R 9q34.13 ND 71 T7 22q12.3 ~1200 72 T9F 21q22.2 ~1600 73 T9 R 22q11.22 ~1600 74 T10 F 7q22.2 ~1300 75 T10 R6q14.1 ~1300 76 T11 F 17q23.3 ~2000 77 T11 R 16q23.1 ~2000 78 T12 4p15.1~2100 79 T13 F 20p13 ~1700 80 T13 R 1p13.3 ~1700 81 T14 R 11q25 ~1500 82T17 2q31.3 ND 83 T18 2q31.1 ND 84 ¹Chromosomal location is determined byBLAT search of DNA sequence data from the STAR elements against thehuman genome database. The location is given according to standardnomenclature referring to the cytogenetic ideogram of each chromosome;e.g., 1p2.3 is the third cytogenetic sub-band of the second cytogeneticband of the short arm of chromosome 1(WorldWideWeb.ncbi.nlm.nih.gov/Class/MLACourse/Genetics/chrombanding.html).F, forward sequencing reaction result; R, reverse sequencing reactionresult. When the forward and reverse sequencing results mapped todifferent genomic locations, each sequence was extended to the fulllength of the original clone (as determined by restriction mapping)based on sequence information from the human genome database. ²ND: NotDetermined.

TABLE 10 Arabidopsis STAR elements of the invention, includingchromosome location and length (SEQ ID NOS: 85-119). STAR ChromosomeLength, kb SEQ ID NO: A1 I 1.2 85 A2 I 0.9 86 A3 I 0.9 87 A4 I 0.8 88 A5I 1.3 89 A6 I 1.4 90 A7 II 1.2 91 A8 II 0.8 92 A9 II 0.9 93 A10 II 1.794 A11 II 1.9 95 A12 II 1.4 96 A13 II 1.2 97 A14 II 2.1 98 A15 II 1.4 99A16 II 0.7 100 A17 II 1.5 101 A18 III 1.5 102 A19 III 0.7 103 A20 III2.0 104 A21 IV 1.8 105 A22 IV 0.8 106 A23 IV 0.6 107 A24 IV 0.5 108 A25V 0.9 109 A26 V 1.9 110 A27 V 1.1 111 A28 V 1.6 112 A29 V 0.9 113 A30 V2.0 114 A31 V 2.0 115 A32 V 1.3 116 A33 V 0.9 117 A34 I 0.9 118 A35 II1.1 119

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1. A cell comprising a first polypeptide expression unit and a secondpolypeptide expression unit, said first and said second polypeptideexpression units each encoding at least one polypeptide of interest,wherein said first and second polypeptide expression units each compriseat least one sequence having the capacity to at least in part blockchromatin-associated repression, wherein the sequence having thecapacity to at least in part block chromatin-associated repression forthe first polypeptide expression unit comprises SEQ ID NO:44 and whereinthe sequence having the capacity to at least in part blockchromatin-associated repression for the second polypeptide expressionunit is selected from the group consisting of any one of SEQ ID NO:1through SEQ ID NO:65.
 2. The cell of claim 1, wherein the sequencehaving the capacity to at least in part block chromatin-associatedrepression for the second polypeptide expression unit comprises SEQ IDNO:44.
 3. The cell of claim 1, wherein said first and second polypeptideexpression units each further encode a different selection marker. 4.The cell of claim 1, wherein at least one of said first and secondpolypeptide expression units comprises a monocistronic gene comprisingan open reading frame encoding a polypeptide of interest and whereinsaid monocistronic gene is under control of a functional promoter. 5.The cell of claim 1, wherein at least one of said first and secondpolypeptide expression units comprises a bicistronic gene comprising inthe following order: (i) an open reading frame encoding a polypeptide ofinterest, (ii) an Internal Ribosome Entry Site (IRES), and (iii) aselection marker, and wherein said bicistronic gene is under control ofa functional promoter.
 6. The cell of claim 1, wherein at least one ofsaid first and second polypeptide expression units comprises at leasttwo of the sequences having the capacity to at least in part blockchromatin-associated repression, arranged such that said polypeptideexpression unit is flanked on both sides by at least one of thesequences having the capacity to at least in part blockchromatin-associated repression.
 7. The cell of claim 1, wherein atleast one polypeptide of interest comprises an immunoglobulin heavychain and the other polypeptide of interest comprises an immunoglobulinlight chain, wherein said heavy and light chain can form a functionalantibody.
 8. A method for expressing at least two polypeptides ofinterest in a cell, said method comprising: providing a cell, the cellcomprising a first polypeptide expression unit and a second polypeptideexpression unit, said first and second polypeptide expression units eachencoding at least one polypeptide of interest, wherein said first andsecond polypeptide expression units each comprise at least one sequencehaving the capacity to at least in part block chromatin-associatedrepression, wherein the sequence having the capacity to at least in partblock chromatin-associated repression for the first polypeptideexpression unit comprises SEQ ID NO:44, and wherein the sequence havingthe capacity to at least in part block chromatin-associated repressionfor the second polypeptide expression unit is selected from the groupconsisting of any one of SEQ ID NO:1 through SEQ ID NO:65; and culturingthe cell under conditions wherein said first and second polypeptideexpression units are expressed.
 9. The method according to claim 8,wherein the sequence having the capacity to at least in part blockchromatin-associated repression for the second polypeptide expressionunit comprises SEQ ID NO:44.
 10. The method according to claim 8,wherein said first and second polypeptide expression units each furtherencode a different selection marker.
 11. The method according to claim8, wherein at least one of said first and second polypeptide expressionunits comprises a monocistronic gene comprising an open reading frameencoding a polypeptide of interest and wherein said monocistronic geneis under control of a functional promoter.
 12. The method according toclaim 8, wherein at least one of said first and second polypeptideexpression units comprises a bicistronic gene comprising in thefollowing order: (i) an open reading frame encoding a polypeptide ofinterest, (ii) an Internal Ribosome Entry Site (IRES), and (iii) aselection marker, and wherein said bicistronic gene is under control ofa functional promoter.
 13. The method according to claim 8, wherein atleast one of said first and second polypeptide expression unitscomprises at least two of the sequences having the capacity to at leastin part block chromatin-associated repression, arranged such that saidpolypeptide expression unit is flanked on both sides by at least one ofthe sequences having the capacity to at least in part blockchromatin-associated repression.
 14. The method according to claim 8,wherein at least one polypeptide of interest comprises an immunoglobulinheavy chain and the other polypeptide of interest comprises animmunoglobulin light chain, wherein said heavy and light chain can forma functional antibody.
 15. A polypeptide expression unit comprising: abicistronic gene comprising in the following order: (i) an open readingframe encoding a polypeptide of interest, (ii) an Internal RibosomeEntry Site (IRES), and (iii) a selection marker, and wherein saidbicistronic gene is under control of a functional promoter; and at leastone sequence having the capacity to at least in part blockchromatin-associated repression, wherein the sequence having thecapacity to at least in part block chromatin-associated repressioncomprises SEQ ID NO:44.
 16. The polypeptide expression unit of claim 15,further comprising a further sequence having the capacity to at least inpart block chromatin-associated repression, wherein said furthersequence is selected from the group consisting of any one of SEQ ID NO:1through SEQ ID NO:65, and wherein said further sequence is arranged withsaid polypeptide expression unit such that said polypeptide expressionunit, on one side, comprises SEQ ID NO:44, and on another side comprisessaid further sequence having the capacity to at least in part blockchromatin-associated repression.
 17. A method for expressing twopolypeptides of interest, the method comprising: a) providing host cellscomprising: (i) a first polypeptide expression unit comprising abicistronic gene comprising a promoter functionally linked to a sequenceencoding a first polypeptide of interest and a first selectable markergene, and (ii) a second polypeptide expression unit comprising abicistronic gene comprising a promoter functionally linked to a sequenceencoding a second polypeptide of interest and a second selectable markergene, wherein said second selectable marker gene is different from saidfirst selectable marker gene, and wherein said first polypeptideexpression unit, or said second polypeptide expression unit, or each ofsaid first and said second polypeptide expression units comprise atleast one sequence having the capacity to at least in part blockchromatin-associated repression, wherein the sequence having thecapacity to at least in part block chromatin-associated repressioncomprises SEQ ID NO:44; b) selecting a host cell by selecting forexpression of said first and second selectable marker genes; and c)culturing a selected host cell to express said first and secondpolypeptides.
 18. The method according to claim 17, wherein each of saidfirst and said second expression units comprise at least one sequencehaving the capacity to at least in part block chromatin-associatedrepression, the sequence having the capacity to at least in part blockchromatin-associated repression comprising SEQ ID NO:44.
 19. The methodaccording to claim 17, wherein the two polypeptides of interest formpart of a multimeric protein.
 20. A set of two polypeptide expressionunits, said set comprising: (i) a first polypeptide expression unitcomprising a bicistronic gene comprising a promoter functionally linkedto a sequence encoding a first polypeptide of interest and a firstselectable marker gene, and (ii) a second polypeptide expression unitcomprising a bicistronic gene comprising a promoter functionally linkedto a sequence encoding a second polypeptide of interest and a secondselectable marker gene, wherein said second selectable marker gene isdifferent from said first selectable marker gene, and wherein said firstpolypeptide expression unit, or said second polypeptide expression unit,or both said first and said second polypeptide expression units compriseat least one sequence having the capacity to at least in part blockchromatin-associated repression, wherein the sequence having thecapacity to at least in part block chromatin-associated repressioncomprises SEQ ID NO:44.
 21. The set of two polypeptide expression unitsof claim 20, wherein both said first and second polypeptide expressionunit have integrated into the genome of a cell.